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%.     

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.     

An assessment of thein vitro germination capacity of pollen grains of five tea hybrid rose cultivars

Summary Germination trials of pollen grains of the tea hybrid rose cultivars: Ferry Porche, Bronze Masterpiece, Queen Elizabeth, John F. Kennedy and Lady X, were carried outin vitro, to determine whether these cultivars are suitable as pollen donors in a breeding programme.Pollen grains of all five cultivars germinated poorly in a medium containing sucrose only. Addition of boric acid in the medium improved the germination percentages. The highest germinability was observed in Ferry Porche and Lady X on 15% sucrose+50 ppm boric acid, and 20% sucrose+100 ppm boric acid, respectively. Addition of calcium nitrate in the medium reduced germination percentages in all cultivars.The effect of pH of the medium on pollen grain germinability depended on the cultivar and the composition of the medium. On a medium free of boric acid, the pH values between 5.5 and 7.0 caused a significant increase in the germination percentages of pollen grains of Lady X and Queen Elizabeth. The addition of boric acid decreased the responsiveness of the pollen of all cultivars to the pH changes of the medium.The germination capacity of pollen grains of Lady X and Ferry Porche was not affected by incubation temperatures ranging between 20°C and 30°C.On the basis of the above results Lady X and Ferry Porche are considered the most suitable for pollen donors.     

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. II. Inheritance of pollen viability

Summary Pollen viability was evaluated in about 500 seedlings originating from 31 crosses between nine commercial Hybrid Tea-rose varieties. The data indicated that pollen viability was inherited additively.     

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.     

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.     

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.     

The production of 2n pollen in rose

Based on the size differences found between haploid and diploid pollen produced by diploid and tetraploid rose cultivars, respectively, 2n pollen producers were identified in a population of 53 diploid hybrids from a cross between a dihaploid rose, derived from the haploidization of a tetraploid modern cultivar and the diploid species R. wichuraiana. Frequency of 2n pollen producers was estimated in 2002, 2003 and 2004. Highly variable frequencies were found i) within population; ii) during years of observation (between years and between different months in the same year). The variation of 2n pollen production could be related to environmental fluctuations. A cytological analysis of male meiosis was carried out in 10 hybrids randomly chosen. Among meiotic abnormalities leading to 2n pollen formation, triads (containing a 2n microspore at one pole and two n microspores at the other) resulting from abnormal spindle geometry were frequently observed. The mode of 2n pollen formation is genetically equivalent to a First Division Restitution (FDR) mechanism. FDR 2n pollen transmits a high percentage of the heterozygosity from the diploid parent – 2n pollen producer-to the tetraploid offspring.     

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     

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     

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     

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.     

Low temperature storage of pistachio pollen

Summary Pollen from four male pistachio (Pistacia vera L.) clones was stored at –196°C and –20°C for up to 12 months and tested for ability to germinate in vitro following a period of hydration at high humidity. Germination of fresh pollen was high (>80%) for each clone. At –196°C, pollen of cv. Peters survived freezing, storage and thawing with no loss of germinability; pollen of the other three clones had sharp declines in germination possibly attributable to cellular lesions incurred during freezing or thawing. When the relative humidity of the –20°C storage environment was maintained at or near 33%, Peters pollen had high rate of germination through 12 months storage. Without control of relative humidity, Peters pollen germination was high at 4 months, but declined at 12 months. Germination requirements became more exacting for pollen stored at –20°C for 12 months at suboptimal humidity conditions. Pollen of the other three clones did not tolerate storage at –20°C as well as Peters pollen regardless of the storage humidity environment.     

Meiosis and pollen grain viability in Helianthus mollis,Helianthus salicifolius,Helianthus maximiliani and their F1 hybrids with cultivated sunflower

Three diploid perennial sunflower species are useful for variety improvement: Helianthus mollis, because of sessile leaves, H. salicifolius, because of a high oil concentration, and H. maximiliani, a potential source of resistance to Sclerotinia sclerotiorum. The crossability of these species to cultivated sunflower was examined.Hybrids were obtained from eight combinations, with 3–15 F₁ plants per combination. The F₁'s exhibited the dominant phenotype of the wild species. Pollen viability varied between 32.1 and 69.9%. Meiosis was irregular in the F₁ hybrids. At diakinesis, bivalents (62.7–97.9% of meiocytes), univalents (0–31.23%), and multivalents (3.84–7.68%) were detected. At anaphase I, chromosome bridges were detected in 6.77 to 11.44% of meiocytes. Fast chromosomes in metaphase I, and lagging chromosomes in anaphase I and telophase II were evidenced in a high percentage of meiocytes.     

Control pollination and pollen management in Sesbania sesban (L.) Merr.

Sesbania sesban was amiable to controlled cross pollination when the emasculation and pollination operations were performed in the morning. A medium of 10% sucrose solution was found to be optimal for in vitro germination of the pollen grains. The pollen is quite tolerant of orthodox storage conditions enabling controlled crosses to be performed among accessions that flower in differing seasons of the year.     

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.     

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.     

Efficacy of organic solvents for storing pollen grains of some leguminous taxa

Summary The efficacy of some organic solvents for storing pollen grains of five leguminous taxa has been assessed and compared with pollen stored under controlled temperature and humidity conditions. The success obtained in the former was invariably less than that in the latter in all the taxa. The results suggest that the use of organic solvents holds some promise only for short-term and not for long-term sotrage.     

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.