Pollen storage of Fragaria and Potentilla

Summary Low temperature and humidity were used for the storage of pollen of four species of Fragaria (2n=14, 42 and 56) and two species of Potentilla (2n=14). The stainability of Fragaria pollen was assessed over a number of years by use of aceto-carmine stain and its viability by cross-pollinations; stainability of Potentilla pollen was assessed by acetocarmine. Fragaria pollen so stored remained stainable for seven years and showed the ability to set seed for three years. over the latter period, aceto-carmine staining gave a reasonable prediction of seed set produced by the stored pollen of Fragaria and so is of value in estimating pollen viability in that genus. Potentilla pollen remained stainable for three years.     

The effect of pollen load and pollen grain competition on fertilization success and progeny performance inFagopyrum esculentum

Summary Flowers of cultivated buckwheat (Fagopyrum esculentum Moench) often receive natural pollen loads of fewer than 10 pollen grains. The cultivated varieties also have high genetic variability. These observations raise the question of whether seed production in buckwheat is often limited by pollen delivery, and whether small increases in pollen load could result in gametophytic selection through pollen grain competition. In greenhouse-grown buckwheat plants, embryo sac penetration by pollen tubes was universal with 10 or more pollen grains. However, seed production increased with pollen load up to 30 grains per flower. Larger pollen loads, which intensify selection among gametophytes, resulted in more vigorous progeny. Seedlings produced from high pollen load (15–20 pollen grains) were larger (40% by weight) than those from low pollen load (5 pollen grains). These results are evidence that pollen grain competition can occur in buckwheat with benefits for progeny performance.     

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.     

Relation between wheat-rye crossability and seed set of common wheat after pollination with other species in the Hordeae

Summary Cultivars of common wheat (Triticum aestivum L. em. Thell.) of high wheat-rye (Secale cereale L.) crossability set more seed with pollen of other related species than did wheats of low wheat-rye crossability. This was found to be true for pollen parents from the genera Triticum, Aegilops, Secale, Agropyron and Elymus.     

Pollination with gamma-irradiated pollen and development of fruits,seeds and parthenogenetic plants in apple

Summary Four apple (Malus X domestica) genotypes, Erovan, Golden Delicious, R1-49 and X6677, were pollinated with a marked pollen irradiated by -rays at doses ranging from 125 to 1000 Gy. Pollination with such irradiated pollen affected fruit set, seed number and seed contents, and induced the formation of parthenocarpic fruits and the development of parthenogenetic embryos. The immature embryos extracted from seeds. 2 and 3 months after pollination, were cultured in vitro and germinated after 2 months of cold treatment (3°C). Haploid plants were obtained in all 4 genotypes, after pollination with pollen irradiated at doses from 200 to 500 Gy. The optimum conditions for induction of apple haploids, by irradiated pollen approach, have been established.     

Wheat pollen dispersal under semiarid field conditions: potential outcrossing with Triticum aestivum and Triticum turgidum

Isolation distance is the main barrier to crop-to-crop gene-flow. A 3-year study assessed the maximum potential outcrossing under field conditions between two wheat cultivars (Triticum aestivum L.) and between wheat and durum wheat (Triticum turgidum L. var. durum). Outcrossing was measured by seed set on emasculated recipient plants placed at four sides with different distances from a 3 m × 3 m T. aestivum (cultivar Chinese Spring) pollen source. Frequencies of seed set at 0 m distance were 45% (37–56%) for T. aestivum cultivars and 18% (5–30%) with T. turgidum. These values agree with hybridization in non-limiting pollen conditions measured by manual crosses in greenhouse. The number of pollen grains and the outcrossing frequencies decreased at increasing distances influenced by the prevailing wind direction. Under semiarid conditions of this assay, viable pollen was found 14 m from the pollen source, with a maximum distance of 8 m at which cross-pollination decreases below 1%. Ambient conditions affect pollen viability, hybridization and pollen dispersal. Data presented in this paper emphasize the major role played by environmental conditions in outcrossing. Data obtained in one area may therefore not coincide with the prevailing situation in different locations and climates.     

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.     

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.     

Influence of timing of cross-versus self-pollination on self-incompatibility in Brussels sprout inbreds

Summary The impact of cross pollination 30 h, 4 h, and immediately prior to self-pollination, and self-pollination 2, 4, 8, 12, 24 and 30 h prior to cross-pollination was assessed for pollen tube number per style, seed number per siliqua, and proportion of self-seed (sibs) per siliqua, in two inbred lines of brussels sprout. Pollination procedure had a marked effect on the amount of sib-and hybrid seed produced. Cross pollination 30 h prior to self-pollination produced significantly greater numbers of sib progeny, as did self-pollination 8–12 h before cross pollination. Prior application of self pollen reduces the number of pollen tubes in the style. The results of this experiment suggest that self-incompatibility in brussels sprout could better be assessed by self-pollination with a subsequent cross pollination 8 h later, and the determination of amounts of sib and F₁ hybrid seed per siliqua.     

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 performance of double or mixed pollinations with compatible and self-incompatible or incongruous pollen of pear and apple

Summary Six pear and five apple trials were carried out to ascertain the outcome of combinations of compatible pollen (C) with self (S) or incongruous pollen (I) as to the pollination index (PI=seeds/pollinated flower). The PI of the mixture C+I (1:5) was consistently depressed as compared to that of the control C. The results of the double pollinations S/C and I/C were affected by the temperature at pollination; their PI's at <15°C were twice as high as those at >15°C, being well above and below the PI of C in the former and latter case respectively. The opposite was true for the C/S combination, the PI of which increased with the pollination temperature; the PI of C/I did not differ much from the PI of C, irrespective of temperature. The conclusion was reached that the interaction previously and presently found between compatible and self-incompatible pollen also exists to a fair extent between compatible and incongruous pollen. However, in pear neither the mentor nor the pioneer pollen technique proved to aid its hybridization with apple, the formation of self seed was not observed either. In apple the production of apple × pear hybrids was likewise doubtful, but the double pollinations S/C and C/S formed 4–10% self seed.     

Estimation of hybrid seed production and efficient pollen flow using insect pollination of male sterile soybeans in caged plots

The aptitude of leafcutter bees to pollinate male sterile soybean plants (ms2 gene) in caged plots was evaluated in four experiments from both quantitative and qualitative points of view. The m.s. plant seed set was satisfactory: on average, it represented 60% of the male fertile isogenic seed set (with a range between 44 and 69%). The lower yield of m.s. plants was linked with a smaller number of fertile reproductive nodes. The efficient pollen flow was observed over the flowering period with both morphological and electrophoretic markers. Insect behaviour was not influenced by flower colour. Differences between flowering duration of pollen donors appear to be the major factor inducing unbalanced populations. The interest of this technique as a tool for dynamic management of the genetic variation in populations or for hybrid seed production is discussed.     

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.     

Effect of selection for pollen grain size on various traits in common bean

Summary Selection among microgametophytes usually exploits variation in pollen grain germination. Studies of variation in pollen grain size in common bean (Phaseolus vulgaris L.) suggested that selection for size might lead to changes in sporophytic traits. To determine whether microgametophytic selection based on size would affect pollen grain size in subsequent generations or sporophytic traits that were correlated with pollen grain size, pollen grains from three crosses were separated into two size categories by sieving and then used to pollinate cv. Diacol Calima. Selection resulted in changes in pollen grain diameter for pollen from F₁, F₂ and F₃ plants for all crosses. In vitro germination indicated no differences between vigor of large and small grains, but extraction and sieving reduced germinability. F₁ seed from two of the crosses with size-selected pollen varied in weight according to pollen grain size, but in subsequent generations, the effect disappeared. Both size categories of selected pollen resulted in F₂ progeny with reduced numbers of seeds per pod as compared to controls, suggesting that the size selection process may have resulted in indirect selection for traits reducing seed set. The overall results suggested that genes determining pollen grain size in bean have little or no effect on sporophytic traits such as seed size and seed yield.     

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.     

Variation amongst protoplast-derived moth bean Vigna aconitifolia plants

Summary Protoplasts were isolated from leaves of a single plant of Vigna aconitifolia, a drought resistant grain legume. The protoplasts regenerated and formed colony and calli from which 50 entire plants were regenerated and transferred to field conditions. Only 7 plants survived upto maturity and they flowered and produced pods with seed. The protoplast derived plants showed variation in important characters. Two groups of characters (one with 7 sets and another with 6 sets) were studied in the protoclones. In first group protoclones showed variations in seed germination, maturity age, pod length, pod and seed colour, number of abortive seed per pod and response to field rots, however, not much difference was recorded in pollen stainability and meiotic behaviour in these protoclones. In second group analysis of variants showed significance difference for plant height, rachis-length, length and breadth of mature odd leaflets, seed per pod and weight of seed. The results indicate that protoplast can be source of variation in this crop. However, detailed biochemical and genetical analysis of protoclones are required.     

Restrictions of the efficiency of haploid plant production in apple cultivar Idared,through parthenogenesisin situ

Summary For haploid plant production in Idared apple, parthenogenesisin situ was induced by irradiated pollen. The most important restrictions were seed set, embryo germination and green plant recovery from germinated embryos. The efficiency of green plant production was strongly influenced by irradiation dose, picking time and quality of the irradiated pollen. 250 Gy pollen was more efficient for green plant production than 500 Gy pollen. Using 250 Gy pollen with reduced germination capacity resulted in a five-fold lower green plant yield. Delaying picking of the fruits (140 instead of 70 days after pollination) had a significant positive effect. Up to 20 plants per 1000 pollinated flowers, showing no expression of a marker gene present in the pollen parent, were obtained in some treatments. Induction efficiency was high when seed weight was high. This offers the possibility of selecting seeds with the best chances for haploid plant production. Heavy seeds generally contained visual normal developed embryos, which germinated better than those from lighter seeds and gave normal plant development. Delayed picking increased the proportion of large seeds, suggesting that pollination with irradiated pollen retards embryo development. From treatments with irradiated pollen of poor quality more small seeds were harvested.     

Pollen grain germination and pollen tube growth following in vivo and in vitro self and interspecific pollinations in annual Cicer species

Summary Germination of pollen grains and growth of pollen tubes were studied to determine the cause of barreness in crosses among annual Cicer species. In vivo and in vitro time-course studies and fluorescent microscopy revealed no pollination incompatibility among the selfs, crosses and reciprocals of C. arietinum L., C. reticulatum Lad. and C. cuneatum Rich. In general, Cicer pollen grains germinated and grew on styles of Cicer species. Pollen tube growth was characterized by irregularly spaced and intermittent callose deposits. Failure of seed formation in interspecific pollinations may be attributed to the slowness of pollen tube growth or collapse of fertilized ovules. In addition to these causes, shortness of stamens and sparsity of pollen grains were responsible for flower drop in natural selfs. Although the number of pollen tubes entering the micropyle in interspecific pollinations was low, it may be possible to grow the fertilized ovules on an artificial medium to obtain F₁ plants.     

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.     

Methods of overcoming interspecific barriers in Trifolium

Summary Barriers to interspecific hybridization in Trifolium were investigated by manipulation of mentor pollen treatments, ploidy levels, and compatibility and male sterility systems. Crosses involving the addition of mentor pollen produced fewer seeds and hybrids than crosses involving normal pollination. Lower seed set with mentor pollen was deduced to result from the use of less viable pollen, approximately half the pollen having been killed by alcohol. Pollinations at the diploid level resulted in more hybrids than at the tetraploid level, perhaps because genes for male sterility produced higher female sterility in the tetraploids. The self-compatible stock produced more seeds, mostly selfs, than the self-incompatible stock, but produced more hybrids only in one cross, T. pratense L. × T. diffusum Ehrh. The use of male-sterile female parents reduced selfing but produced fewer hybrids than male-fertile female parents. Techniques of this study were designed to affect prefertilization barriers, but the lack of effect may indicate that postfertilization barriers in Trifolium are of greater importance.Journal Article No. 98-3-208 of Kentucky Agricultural Experiment Station. Published with approval of the Director.