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.     

Investigations into the Barrier(s) to Interspecific Hybridization between Cicer arietinum L. and eight other Annual Cicer species

Barrier(s) to interspecific hybridization between the cultivated chickpea, Cicer arietinum L., and eight other annual wild species, i.e. C. reticulatum Lad., C. echinospermum Dav., C. pinnatifidum J. and S., C. judaicum Boiss., C. bijugum Rech., C. chorassanicum (Bge) M. Pop., C. yamashitae Kit. and C. cuneatum Rich., were investigated. In general, good pollen germination and pollen tube growth were observed in all eight crosses and their reciprocals. En spite of a few pollen cube growth abnormalities in most crosses, pollen tube penetration into the ovule and, thus, fertilization was observed in all cross combinations. However, differences were observed in the time from pollination to fertilization, not only between different interspecific crosses but also between reciprocals of a particular interspecific cross. The crossability barrier is, therefore, believed to be due to factor(s) operating after fertilization.     

Overcoming unilateral breeding barriers between Lycopersicon peruvianum and cultivated tomato,Lycopersicon esculentum

Summary Treatment of Lycopersicon peruvianum stigmas with an artificial medium analogous to stigmatic exudate allowed pollen germination and growth on immature pistils. Growth of Lycopersicon esculentum pollen tubes to L. peruvianum ovules, an otherwise incompatible cross, was achieved following such treated bud-pollinations. No plantlets were recovered, although a few embryos from this cross at the globularity heart stage of development were excised at 22 days after pollination, indicating the presence of crossing failures as severe as in the reciprocal cross. Hybrid plants were obtained from the reciprocal cross, using as pollen parent an L. peruvianum line selected for congruity with L. esculentum. Bud pollinations to L. peruvianum, using these interspecific F₁ hybrids as the pollen parent, allowed viable embryo development and plantlet recovery. Resulting backcross plants, which possess approximately 1/4 L. esculentum genome in a L. peruvianum cytoplasm, may facilitate further introgression of the L. esculentum nuclear genes into in foreign cytoplasm.     

Factors affecting haploid production in wheat using the Hordeum bulbosum system. 1. Genotypic and environmental effects on pollen grain germination,pollen tube growth and the frequency of fertilization

Summary A quantitative examination of pollen grain germination and pollen tube growth within the overy wall was made in selected wheat × tetraploid H. bulbosum hybridizations, to investigate the effect of the wheat and the H. bulbosum genotype on these characters. As expected, variation at the known crossability loci had no effect on pollen grain germination. The frequency of pollen tube penetration of the ovary wall was, however, severely reduced when the dominant alleles were present. Pollen tube penetration was nevertheless observed in 3 of the 110 ovaries examined of the non-crossable cultivar Highbury. The H. bulbosum genotype had a much smaller effect on these characters, but significant differences between the clones were observed in the numbers of pollen tubes initially penetrating the ovary wall. Although two H. bulbosum genotypes showed no significant differences in the number of ovaries with pollen tubes at the base of the ovule, significant differences in the frequency of fertilization were observed. The possible cause of this discrepancy is discussed.The frequency of fertilization in crossable wheat × H. bulbosum hybridizations was improved by the application of gibberellic acid within 10 minutes of pollination, and reduced by an increase in the ambient temperature from 20°C to 26°C. Fertilization following the pollination of non-crossable wheat genotypes was not affected by either of these factors.     

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 tube growth and fertilization after different in vitro pollination techniques of Aechmea fasciata

The effect of cut style and placental pollination on fertilization efficiency was studied for compatible Aechmea fasciata plants. These alternative in vitro pollination techniques resulted in lower penetration rates of the ovules by pollen tubes in comparison to pollination on the stigma. An explanation was found in the intervention of the normal pollination process,through which less pollen-pistil interactions were built up. After cut style pollination the percentage of ovule penetration by pollen tubes increased when a longer style part was left at the ovary. Probably fewer factors that control pollen tube growth are present in the lower style part. Pollen germinated on the ovules after placental pollination but only rarely penetration of the micropyle by a pollen tube occurred. Activation of the ovary, induced by in vivo prepollination for 6 hours, and pollination two days after anthesis did not increase the fertilization percentage. Grafting a style with active growing pollen tubes to ovules on the placenta (placental grafted style pollination) resulted in a higher fertilization percentage. Pollen tube growth through the style was essential for pollen tube guidance to the ovules and penetration of the micropyle. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of mentor pollen on pistil-pollen incongruities among species of Cucumis L.

Summary The merits of mentor pollen in certain interspecific hybridizations in the genus Cucumis L. were evaluated. African wild cucumber species Cucumis metuliferus Naud. and C. africanus L. and Asian species C. sativus var. hardwickii Alef and var. sikkimensis Hook, were reciprocally crossed. Pollen tube growth was arrested halfway down the style in all combinations except in C. sativus × C. africanus. Mentor pollen irradiated with 100 and 200 krad grew through the style and into the ovules in all species. Fruitset was obtained in all cases when mentor pollen was used either alone or in mixtures. Fruits set after self pollination with mentor pollen yielded only seeds without embryos.In several crosses, aided by mentor pollen, a number of relatively large seeds (with enlarged embryosac) were obtained. A sample of these embryosacs contained a globular structure like an embryo. Embryosacs with embryo-like structures were explanted on several different media but no development was obtained.     

The crossing of common wheat (Triticum aestivum L.) with cultivated rye (Secale cereale L.). II. Fertilization and early post-fertilization developments

Summary Fertilization and early seed development was studied in the variety Chinese Spring of common wheat (Triticum aestivum L.) after pollination with rye (Secale cereale L.) and selfing, and in the common wheat variety Hope after selfing. In all three combinations the first pollen tube reached the micropyle in about 40 min after pollination. When pollinated with rye the migration of the sperm nuclei to the egg cell and the polar nuclei was delayed by about an hour. In the subsequent development until 72 hours after pollination the average cellular and nuclear doubling times of embryo and endosperm were about 16 and 8 hours for the interspecific combination. 18 and 9 hours for Chinese Spring selfed and 20 and 12 hours for Hope selfed.     

Pollen germination,pollen tube growth and fertilization following self and interspecific pollination of Paspalum species

Summary Crossability between most Paspalum species is very low. This study was undertaken to identify the impediments to hybridization. Accessions of P. intermedium Munro. ex Morong, P. jurgensii Hackel and P. dilatatum Poir were self-pollinated and crossed with one anther. Paspalum intermedium is essentially self-sterile but P. jurgensii and P. dilatatum are highly self-fertile. Following pollination, pollen germination and tube growth were studied by observing the pollinated pistils with fluorescent microscopy. Examination of self-pollinated pistils revealed that the pollen germinated shortly after contacting the stigmas. Germination was over 80% for the P. intermedium and P. dilatatum accessions but only 57% for P. jurgensii. Pollen tubes grew to the micropyle within 45 minutes after pollination in P. dilatatum and 1 hour and 15 minutes in P. jurgensii. However, in the P. intermedium accessions most tubes did not grow beyond the stigma and very few penetrated the style and ovary. Apparently stylar-incompatibility is the reason for the low selfed seed set. In the cross-pollinations, pollen germinated shortly after pollination and germination ranged from 57 to 88% for the different crosses. In all crosses the pollen tubes grew to the micropyle within 30 minutes to 2 hours after pollination indicating that a cross-incompatibility system is not the cause for low crossability among these species. By examining embryo sacs from P. intermedium × P. dilatatum, its reciprocal and P. intermedium × P. urvillei crosses, it was determined that gametes failed to unite in some crosses and this is a major reason for low crossability.     

Effect of humidity on intergeneric pollinations of iap (Inhibition of Alien Pollen) sorghum [Sorghum bicolor (L.) Moench]

The production of interspecific or intergeneric progeny involving Sorghum spp. is greatly enhanced by the presence of the iap (Inhibition of Alien Pollen) allele in sorghum [Sorghum bicolor (L.) Moench]. Variation in the expressivity of the iap phenotype has been observed in previous studies, leading to the hypothesis that the humidity during pollination is affecting the degree of alien pollen adhesion and pollen tube growth. The objective of this study was to determine the optimum humidity for maximum maize pollen tube growth on sorghum florets homozygous for iap. Growth chambers were used to create environments of 45, 65, and 85 % humidity with reciprocal crosses made between the 65 and 85 % humidity chambers. Maize pollen was dusted onto the stigmas of Tx3361 (iap iap) and ATx623 (Iap Iap). Pollen adhesion and germination were higher for pollinations with Tx3361 at all humidity levels but the greatest adhesion and germination was observed at 45 % humidity for Tx3361. Under this humidity, pollen tube growth to the style and ovary was observed. The results indicate that lower humidity maximizes maize pollen adhesion and germination on the stigmas of Tx3361. Analysis of the differences in adhesion between Tx3361 and ATx623 indicate that Iap affects pollen adhesion at the stigma surface and may be controlling pollen–pistil incompatibility in interspecific crosses in sorghum.     

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.     

Independence of self-compatibility and potentiality for self-pollination in peach x almond hybrids

Summary The almond of commerce (Prunus dulcis (Mill.) D.A. Webb) is self-incompatible (SI) and requires honey-bees to effect the transfer of pollen among cultivars that flower simultaneously. Four year old trees from the F₂ generation of several peach x almond hybrids were studied to determine whether self-compatibility (SC) and the potentiality for natural, i.e., abiotic, self pollination (NSP) are genetically related or are inherited independently. Both SC and the high potentiality for NSP are characteristic of peach (Prunus persica (L.) Batsch) but not almond. Forty percent of SC genotypes exhibited adequate NSP (SC, +NSP) for good fertility i.e., without insect-mediated pollination. The remaining 60% of SC genotypes (SC,-NSP) exhibited an average 61% reduction in fruit set on limbs bagged to exclude honeybees during anthesis relative to fruit set on open pollinated limbs. Our data are consistent with the concept that fertility is dependent upon the load of compatible pollen deposited on the stigma. Fruit set reduction on bagged limbs, compared with bagged and self-pollinated limbs, was presumably due to a) lack of/insufficient pollination for fertilization and/or b) post-zygotic abortion of genetically inferior recombinants. Selection following manual self-pollination may result in SC genotypes with or without the capacity for NSP. In contrast, significant fruit set on limbs enclosed during pistil receptivity necessitates that the genotype selected express both SC and the potentiality for NSP.     

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.     

Pollen–pistil interaction in the inter-specific crosses of Sesamum sp

The wild species of Sesamum have long been recognized as an important source of many valuable pest and disease resistance genes and other novel traits such as male sterility and plant type for the cultivated taxon. Utilization of these species in crop improvement is marginal because of their inherent low crossability with the cultigen. An understanding of the biological nature of incompatibility systems that impede hybridization offers tools for successful introgressions into cultivated sesame. The objective of this investigation was to observe pollen germination and pollen tube growth in cross pollinated S. indicum pistils as an indicator of pre-zygotic barriers operating in wide crosses involving S. indicum. Three wild species: S. alatum, S. radiatum and S. laciniatum were used as pollen donors in hybridization with S. indicum. In the cross S. indicum × S. alatum 85.23% of pollen grains germinated and pollen tubes reached ovaries within 8 h after pollination. In other two crosses, S. indicum × S. laciniatum and S. indicum × S. radiatum the level of incompatibility was high as evidenced by a reduced pollen germination and slow rate of pollen tube growth. Measures to circumvent incompatibility in these crosses to produce interspecific hybrids are discussed.     

Optimal pollination conditions for seed set after a self-pollination,an intraspecific cross and an interspecific cross of marrow-stem kale (Brassica oleracea var. acephala)

Summary The effects of different pollination techniques, with and without emasculation and delayed pollination, were investigated to find the conditions for maximum seed set after self-pollination and intraspecific and interspecific crosses of Brassica oleracea var. acephala. The results indicated that the pollination conditions achieving maximum seed set vary with the type of pollination. After controlled self-pollination, the best seed set occurs in bud 3 to bud 10. For the intraspecific cross, the youngest flower and the oldest bud produced the largest number of developed ovules but bud pollination was productive to bud 8. The yields from these two pollination types were best when the female parent was not emasculated. In the interspecific cross with B. campestris cv. Marco the best results came from the youngest flowers and the oldest buds subjected to the standard practice of pollinating directly after emasculation. Possible reasons for these effects are discussed.     

Increasing resistance to Fusarium crown and root rot in asparagus by gametophyte selection

In garden asparagus, Fusarium crown and root rot is the main cause of crop decline. Since chemical treatments are inefficient, efforts should focus on the development of resistant cultivars to control the disease. Toxic culture filtrate (TCF) of F. oxysporum has affected asparagus pollen germination and tube growth. Consequently, gametophyte selection was evaluated to ascertain if the application of selective agents at this level could increase selection efficiency. Two susceptible pistillate plants and one tolerant and one susceptible staminate plants were used in controlled crosses. Before pollination, a drop of a germination vehicle with TCF or without it was applied to the stigmas. Some pollinated pistils were fixed and analyzed by fluorescence microscopy; the rest were left on the plant for seed production. Fifty to 200 seeds were obtained per treatment combination (staminate plant x pistillate plant x pollination vehicle). The derived plantlets were inoculated in vitroand evaluated for disease symptoms. The application of TCF to stigmas reduced pollen germination and tube growth compared with untreated controls,regardless of the genotypic combination. Pollen germination and tube growth was poorer for the tolerant staminate genotype than for the susceptible one. When the TCF was applied, the number of seeds per pollination in comparison with the controls diminished only when the susceptible genotype was the pollinator. The percentage of affected root area of the progenies obtained after applying the TCF was lower than in the controls only when the tolerant genotype was the pollinator. Increasing Fusarium resistance in asparagus by means of gametophyte selection seems feasible. This revised version was published online in August 2006 with corrections to the Cover Date.     

Results of multiple pollination in blueberry (Vaccinium elliottii Chapm.)

Blueberry (Vaccinium section Cyanococcus, family Ericaceae) is a shrub that produces multiple-seeded berries in which only a fraction of the ovules develop into viable seeds. A recessive yellowleaf marker gene was used to evaluate the efectiveness of a single pollination versus multiple pollinations in producing seeds in Vaccinium elliottii Chapm. A multiple-pollination experiment was conducted to see if the first pollen applied or the second produced more progeny, and to see if multiple pollinations increased the number of seedlings produced. Flowers of diploid yellowleaf Vaccinium elliottii were pollinated from one to four times at daily intervals. Pollen from redleaf and yellowleaf plants was used to produce two types of seedlings that could be distinguished visually. Generally, percent berry set, seeds per berry, and seed germination were not increased by multiple pollination. After multiple pollination, the pollen applied first always produced the most seedlings. Prior pollination greatly reduced seed set from subsequent pollination, but subsequent pollination caused little or no decrease in the number of seeds set by the first pollination. This revised version was published online in July 2006 with corrections to the Cover Date.     

Interspecific Hybridization in the Genus Lens Applying in Vitro Embryo Rescue

Summary Four different hybridization experiments were carried out to obtain interspecific hybrids with Spanish cultivars of lentil (Lens culinaris M.). In hybridization experiments I and II, undertaken with only pollination and pollination with the addition of gibberellic acid after fertilization, respectively, no lentil hybrids were recovered. A single interspecific hybrid with L. odemensis was obtained in experiment III using the embryo rescue protocol of Cohen et al. (1984), in this case, a crossing efficiency of 0.11% and a rescue efficiency of 2.5% were obtained. Hybridization experiment IV used a specific embryo rescue protocol developed in this study. In this experiment, ovule-embryos of 18 DAP were cultured on MS salts with 1% sucrose and 1 μ M IAA + 0.8 μ M KN; after two weeks, embryos were released from the ovular integuments and cultured on the same medium for another two weeks in upright position. Afterwards, the embryos were transferred to test tubes containing the same medium and one month later plantlets were obtained. Using the above protocol, out of a total of 1707 pollinations, 6 interspecific hybrids with L. odemensis, 2 with L. nigricans and one with L. ervoides were recovered, yielding on average a crossing efficiency of 0.53% and an average rescue efficiency of 8.26%. Taking into consideration only the interspecific crossing blocks in which hybrids were recovered, the crossing efficiency with L. odemensis was 9%, while with both L. nigircans and L. ervoides the crossing efficiencies were 3%. Rescue efficiencies based on hybrids recovered per number of ovules cultured ranged between 50–100%.     

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.     

Use of Vaccinium octoploids to facilitate 4x-6x gene transfers

The use of an 8x Vaccinium produced through doubling a tetraploid hybrid with colchicine was studied as a means of facilitating gene exchange between V. corymbosum L. and V. ashei Reade. Analysis included meiotic observations and crossability studies of the 8x plant, as well as attempting to develop 6x breeding lines. Meiotic analysis revealed the presence of micronuclei in Telphase II products and polyspory in sporads. In the crossability studies five 4x highbush plants were pollinated with 8x o2 4x pollen. Data gathered included: % fruit set, average weight/fruit, average number of seeds/fruit, average number of seedlings/pollination, and number of 6x seedlings. Octoploid pollinations were significantly lower than 4x pollinations in all parameters. One 6x plant was produced from the 4x-8x crosses but was found to be mitotically unstable, having somatic cells with chromosome numbers ranging from 48 to 168.