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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Techniques for collecting,handling germinating,and storing of pollen of the filbert (Corylus spp.)

Summary Temperature and humidity immediately before dehiscence were important in maintaining viability and germination of filbert pollen. Air temperatures at or above 23°C during dehiscence and pollen storage resulted in nonviability after 8–24 h. Cut branches bearing elongating catkins when exposed to temperatures of 18°±2°C during dehiscence yielded viable pollen. Manganese and boron added to the medium did not significantly increase germination, while 10–100 p.p.m. boron appeared to inhibit it. Storage tests indicated that filbert pollen viability may be maintained at 92% relative humidity and –18°C for at least 12 months.Also published as Tech. Paper No. 2305, Oregon Agricultural Experiment Station.     

The viability and storage of strawberry pollen

Investigations were carried out in 1990 and 1991 to estimate the pollen viability of five strawberry genotypes and their suitability for storage. Pollen viability was assessed by acetocarmine staining, in vitro germination, and in vivo assays. Pollen was stored in darkness at ?18°C for 1 year, and pollen viability was estimated every 4 months during storage. The highest percentage of stained and in vitro germinated pollen grains, respectively, was shown by fresh pollen of cv. ‘Dukat’ (91 and 45%). The lowest values of these characteristics were observed in pollen of cv. ‘Paula’ (48% and 20%, respectively). The best response to storage at ?18°C occurred in pollen of the breeding clone ‘B-302’ and the cv. ‘Redgauntlet’. ‘Paula’ pollen responded least favourably to this. Pollen of the cvs. ‘Dukaf’. ‘Senga Sengana’, and clone ‘B-302’ after storage for 4 months, still had sufficient capacity for fruit set after cross-pollinations.     

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.     

Pollen storage in cocoyam (Xanthosoma sagittifolium (L.) Schott)

Summary The effect of different storage conditions on cocoyam pollen viability was investigated. Two levels of temperature (0° and 5°C) and four levels of relative humidity (0, 10, 20 and 30 percent) represented the storage environments.Viable cocoyam pollen could be obtained after 28 days in storage. The best storage condition was 5°C and 30% relative humidity.     

In vitro pollen germination and tube growth of tomato (Lycopersicon esculentum Mill.) and its relation with plant growth

Summary In vitro pollen germination at 10°C, 14°C and 22°C of four groups of two pure line tomato varieties was compared with their plant growth at 19°C D/10°C N under controlled environmental conditions. Generally, pollen germination was slow at 10°C but after 6 h the percentage of germination was similar to that at 22°C. Maximum germination was obtained at 14°C already after 4 h. The longest pollen tubes occurred at 22°C. The two varieties within each group differed significantly from each other for percentage of pollen germination. For one group, this difference was greater at 10°C than at 22°C. The varieties in two groups also differed significantly for pollen tube growth. These differences in pollen tube growth rate were greater at 22°C than at 10°C. There was no clear relationship between pollen germination, pollen tube growth and plant growth in any of the four pairs of varieties. The results are discussed with regard to the possibility of pollen selection at low temperature in order to improve the efficiency of breeding for growth at low temperature.     

Effects of high temperatures on mature pollen grains in wild and cultivated maize accessions

Summary The effects of high temperature on mature pollen of various maize lines were investigated. Genotypic differences in pollen reaction to high temperature were revealed. Pollen grains resistant to high temperature (35°C, 26°C) were characterized by higher germination capacity and better ability to develop normal pollen tubes. The studies are of interest to evaluate reproductive system tolerance and conduct gamete selection at the mature pollen grain stage in maize.     

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.     

Effect of pre-incubation humidity and temperature treatment on the in vitro germination of avocado pollen grains

Optimum in vitro germination of pollen grain of the avocado cultivars Fuerte, Nabal, Ettinger, Bacon and Zutano occurred at 25 °C. However, there were significant differences between cultivars in percentage germination and relative humidity (RH) requirements for optimum pollen grain growth. The most sensitive cultivar to relative humidity was Fuerte, in which the germination of pollen grain rose from 11.4%, at 40% RH, to about 50%, after one hour at 100% RH. The germination% of Nabal pollen grain was already high at 40% RH and was not increased by higher relative humidity. Increased relative humidity also helped to sustain the viability of avocado pollen. At 30 °C and 5% RH the pollen grains of Fuerte quickly lost its ability to germinate, at 40% RH for 1 hour, germination was reduced spectacularly compared to pollen kept in saturated with moisture environment where it was not affected the first 24 hours. The effects of temperature and relative humidity on fruit-set and yield of avocado are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Pollen and pollination experiments. III. The viability of apple and pear pollen as affected by irradiation and storage

Summary Apple and pear pollen was irradiated with doses of 0, 50, 100, 250 and 500 krad (gamma rays) and stored at 4°C and 0–10% r.h. From the in-vitro germination percentages an average LD 50 dose of about 220 krad was estimated. For both irradiated and untreated pollen a close and corresponding lineair relationship existed between germination percentage and pollen tube growth.Irradiated pollen was much more sensitive to dry storage conditions than untreated pollen, resulting in less germination and more bursting. Apparently, irradiation caused the pollen cell membrane to lose its flexibility faster than normal. Rehydration of dry-stored, irradiated pollen in water-saturated air restored germination percentages up to their initial levels. The importance of this procedure in germination trials is stressed.     

Differential cold sensitivity of pollen grain germination in two Prunus species

Summary Pollen grains from selected cutivars of almond [Prunus dulcis (Mill.) D. A. Webb] and peach (Prunus persica Batsch L.) were exposed to a range of temperatures between 1°C and 34°C on a thermogradient plate. Pollen germination at temperatures below 9°C was conspicuously greater in almond than peach. Miximal germination percentages were attained at about 16°C (almond) and 23°C (peach). The two species did not differ in their capacity for pollen tube elongation over a broad range of temperatures. Maximal pollen tube elongation occurred at temperatures 5°C to 8°C higher than maximal pollen germination. Species affiliation appeared to be of much greater consequence than chilling requirement or bloom date of the sporophyte in predicting gametophytic response to temperature.