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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Pollen longevity and artificial cross-pollination in Coffea arabica L

Summary Various aspects of pollen longevity, in vitro germination of pollen and controlled pollination in Coffea arabica were investigated. High pollen viability could be maintained for more than two years, much longer than previously reported, by storing it under vacuum at 18°C. The most satisfactory method of in vitro germination of pollen was the hanging drop in a Van Tieghem cell, with a 10°, sucrose solution. For artificial cross-pollination it is necessary to carry out emasculation and bagging at the latest one day before anthesis. The stigmas of unpollinated flowers remained receptive for at least nine days.This paper is published with permission of the Director, Coffee Research Station, Ruiru, Kenya     

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.     

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.     

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.     

The breeding systems and some interspecific relations of a number of African Trifolium spp.

The breeding systems of 12 species and varieties of Trifolium from Africa were investigated. One annual and the three perennial species were allogamous and self-incompatible, the other annuals were autogamous.The effect of temperature on pollen germination and growth was variable. In some species high temperatures (30°C) adversely affected pollen germination and low temperatures (20°C) in others. A high humidity (93–98% R.H.) was essential for good pollen germination and growth.Approach grafts indicated that these species were closely related, however, this method of grafting may not be as reliable as the cleft graft for assessing species relations. From hybridization attempts between seven species, it appears that some interspecific hybrids may be possible within this group.     

In vitro interspecific fertilization,embryo development and formation of hybrid seedlings between Gossypium hirsutum and G. arboreum

Summary In vitro hybridization between Gossypium hirsutum, and G. arboreum was carried out. Hybrid seedlings were obtained after successive use of the following five kinds of media: 1) pollen grain germination medium, 2) double-fertilization medium, 3) embryo development medium, 4) seedlings formation medium, 5) green seedlings growth medium. The factors affecting in vitro interspecific fertilization, embryogenesis and seedling formation were studied. The key factors were temperature and relative humidity (RH). The optimum RH for in vitro interspecific fertilization was 65%, and a suitable temperature range was 26–30°C. Pollen grain germination ratio decreased rapidly at a RH of 80%. When the temperature and the RH were higher than 32°C and 80%, respectively, the fertilization rate decreased to zero.Effects of petals and calyces of the maternal flowers on in vitro interspecific fertilization and ovule growth were identified. Even though the petals or calyces were excised, hybrid plantlets were also obtained after media were improved and a shake culture was used. In addition, the process of double-fertilization and embryo development were studied cytologically. The developmental characteristics of the hybrid embryos derived from in vitro interspecific fertilization were a later occurrence of the double-fertilization process and a much lesser division of endosperm cells. But the embryo development was not affected by these characteristics, and young hybrid embryos can develop to their cotyledon stage finally on artificial media.     

Studies on sexual variation in mulberry (Morus spp.)

Summary Sex expression in mulberry (Morus spp.) was recorded in 301 varieties, collected from diverse geographical origins, evolved varieties and polyploids maintained at Central Sericultural Research and Training Institute, Berhampore, West Bengal. India (24°6'N and 88°15'E) during 1991–93. Out of total varieties, 49 (16%) male, 161 (53%) female, 52 (17%) monoecious and 39 (13%) were bisexual. Parameters on flowering time, anthesis, floral characters, sex expression and sex reversal were recorded. The indigenous, evolved and polyploids showed early flowering (Jan–Feb) and exotic showed late flowering (Jan–April). The pollen grain viability, seed setting % and other floral behaviour were recorded.     

Overcoming the self-incompatibility of Lilium longiflorum Thunb. by application of flower-organ extract or temperature treatment of pollen

Summary The application of flower organ extracts to stigmas and the temperature treatment of pollen were tried to overcome self-incompatibility of Lilium longiflorum cv. Georgia.Substances in stigma, style, ovary and anther were extracted with ethanol and fractionated with ethylacetate into the acidic, basic and aqueous fractions. The extracs melted in a small volume of distilled water were applied to stigmas prior to self-pollination. Hinomoto stigma extract, self-pollinated and cross-pollinated Georgia stigma extracts of high concentrations and Georgia anther extract of high concentration were effective in overcoming the self-incompatibility and resulted in a high percentage of fruit set and many normal seeds. Extracts from Hinomoto ovary, style and anther were ineffective, except a basic fraction, which was very slightly effective.Pollen was treated with 40°C for 60 or 90 minutes and 50°C for 30 or 60 minutes, and a half of each lot was followed by –20°C for 24 h, prior to self-pollination. All treatments were effective, especially at 40°C for 60 minutes or 50°C for 30 minutes, and 40°C for 90 minutes or 50°C for 60 minutes followed by –20°C for 24 h.     

The breeding system of rubber (Hevea brasiliensis): an evaluation of controlled hand pollination methods

Summary Controlled hand pollinated pistils of rubber were observed using fluorescence microscopy to assess the efficiency of the universally-employed method for the production of progeny for plant breeding. The controlled hand pollination method conducted in the morning resulted in the deposition of a mean of 15.6 pollen grains on the stigma, with no stray pollination observed. Over 36% of the pistils had the potential to set fruit.Pollinations conducted in the afternoon at the normal time of anthesis, had double the fruit set potential of morning pollinations as measured by penetration of ovules by pollen tubes. Pollinator efficiency also varied, with excessive damage to stigmas resulting in reduced pollen germination and tube growth. There were differences between clones in both female and male fertility, in the proportion of pistils with more than three carpels and in the production of small abnormal stigmas. There was no difference in pollen tube growth following self- or cross-pollination, indicating that the self-sterility mechanism of rubber operates in the ovary. Pollen could be stored for 5 days at 5°C and 75% RH with a 22% loss of fertility.