Effects of gamma irradiation on in vitro pollen germination of different Cucumis species

Summary Pollen grains of 14 Cucumis accessions were irradiated with 0, 1, 2, 3 or 4 kGy acute gamma rays and germinated in vitro directly afterwards. Pollen germination was significantly reduced by increasing irradiation dose for all species, except C. melo var. agrestis. Pollen tube growth was generally reduced likewise. Pollen of two C. anguria subspecies was most sensitive to irradiation. Sensitivity of the pollen with respect to pollen tube growth in relation to irradiation dose was inversely related to total DNA amount per nucleus. In vitro germination was not related to DNA amount per nucleus. Results show that the examined Cucumis species, especially C. melo var. agrestis, are sufficiently resistant to irradiation to be used as donor species for in vivo egg cell transformation of the cucumber.     

Chromosome studies in the genus Cucumis

Summary Cytological investigations of 50 wild Cucumis introductions revealed the presence of three tetraploid species with 2n=48 chromosomes, and one hexaploid species with 2n=72 chromosomes, while all other species are diploid containing 2ns=14 or 2n=24 chromosomes. Two of the tetraploid species, C. heptadactylus (P.I. 282446), and a species related to C. zeyheri (P.I. 273192, 299570, 299571, and 299572) are natives of South Africa, while C. aculeatus (P.I. 193967, 196844, 273648, 273649, and 273650) is found in Ethiopia. The hexaploid, C. figarei (P.I. 343699, 343700, 343701), is a native of Nigeria. All polyploids are perennial, have efficient vegetative reproduction systems, and may have originated from the spontaneous formation of polysomatic cells.Supported by the Colorado State University Experiment Station and published as Scientific Series Paper No. 2089.     

Interspecific hybridization in Cucumis L. II. The crossability of species,an investigation of in vivo pollen tube growth and seed set

Summary The crossability of 12 Cucumis species of African and Asiatic origin was studied in a diallel cross, in order to find ways to realise the cross between the common cucumber (C. sativus L.) and its wild relatives which carry resistances against diseases and pests.Self-pollinations and cross-pollinations within species gave normal pollen tube growth and seet set. The different accessions within a species, as a rule reacted alike in interspecific crosses. In crosses between African species different crossing patterns were found, viz. bilateral congruity, bilateral incongruity and unilateral incongruity. Within C. sativus all accessions intercrossed freely, except one, which displayed unilateral incongruity.Good seed was harvested from several crosses and in some cases embryo culture was needed for further development of seeds. No good seeds were obtained from any cross between a species of the African group and C. sativus L.     

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.     

Synthesis and preliminary characterization of a new species (amphidiploid) in Cucumis

A successful interspecific hybridization between cucumber (Cucumis sativus L., 2n = 14) and a wild it Cucumis species, C. hystrix Chakr. (2n = 24) was made via embryo rescue. Hybrid plants (2n = 19; 7 from cucumber and 12 from C. hystrix) were sterile, but morphologically uniform. Self-pollination and backcrossing of F₁ hybrid plants to either parent confirmed presence of both male- and female-sterility that were likely caused by lack of homology and improper pairing during meiosis. While the multiple-branching habit, densely brown hairs (on corolla and pistil), orange-yellow corolla, and ovate fruit of F₁ hybrid plants were similar to that of the C. hystrix parent, the appearance of the first pistillate flower was more similar to that of C. sativus parent. The diameter and internode length of the stem, and the shape and size of leaves and flowers were intermediate when compared to the parents. The chromosome number in the hybrid was doubled through somaclonal variation during embryo culture and regeneration process to restore the fertility. Pollen grains were released and fruits with viable seeds matured on fertile, synthetic amphidiploid plants. The results from flow cytometry indicated that, on average, 7.3% of the morphologically unique regenerants had the 4C DNA content of 2.35 pg relative to the 2C DNA content of the original F₁ hybrid at 1.17 pg and, therefore, were likely chromosome-doubled F₁ hybrids (2n = 38). Nutrition alanalysis indicated that the synthetic species had higher protein (0.78%)and mineral (0.35%) content compared to the normal pickling cucumber(0.62% and 0.27%, respectively), and could be considered a new Cucumis crop having a special place in the future agriculture. Preliminarily evaluation indicates that C. hystrix possesses a high level of root-knot nematode resistance, and that this resistance is partially expressed in the interspecific F₁ and chromosome-doubled F₁. This and the fact that the fruit morphology of the fertile amphidiploid differs during the growing season (e.g., short and long fruit) suggest that it could be useful in broadening the germplasm base of cucumber. This revised version was published online in July 2006 with corrections to the Cover Date.     

Occurrence of 2n and jumbo pollen in the Brazilian ginseng (Pfaffia glomerata and P. tuberosa)

An analysis of microsporogenesis was carried out in four populations of Pfaffia tuberosa (2n=4x=68) and in ten populations of P. glomerata(2n=2x=34). Monads, dyads and triads were reported at high frequency in all populations of P. tuberosa. In P. glomerata, on the other hand, only four populations presented monads, and at low frequency. Dyads and triads were observed in both species, albeit at a higher frequency in P. tuberosa populations. Certain abnormalities of spindles in the second division, such as sequential and tripolar spindles, led to triad formation by the rejoining of two chromosome sets. The precise causes of monad and dyad formation have not been found during meiosis, although evidence exists that dyads originated by omission of second meiosis and monads by omission of both meiotic divisions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Estimating pollen fertility in Solanum species and haploids

Summary Three staining methods (acetocarmine, fuchsin and oxidation of benzidine) and germination in vitro and in vivo were applied to estimate pollen fertility in Solanum species and dihaploids. Pollen was divided into six classes based on shape and contents of the grains. With acetocarmine, fuchsin, peroxidase and germination in vitro 4, 3, 2 and 1 classes, respectively, are supposed to be included in the percentage of good pollen as measured by these methods. This percentage therefore, in more than 96% of the cases studied, shows a decrease in the order indicated. Neither aging of pollen at room conditions nor collecting pollen from flowers on 1–9 days after anthesis does influence the percentage of good pollen with acetocarmine and fuchsin, whereas this percentage drops sharply to zero with peroxidase and germination in vitro. The latter two methods apparently measure as good pollen only the grains with living cytoplasm. When pollen is collected at three successive dates from the same flowers the percentage of good pollen drops sharply with all methods used. There is a relation between quantity of pollen per flower and pollen quality (% good), low-quantity pollen containing significantly lower percentages of good pollen than medium- and high-quantity pollen. The latter two are not significantly different in this respect.From calculations of correlation coefficients it is concluded that only germination of pollen in vitro is significantly correlated with berry and seed set and thus gives a reliable estimate of male fertility. This does not hold true for the two staining methods without due reserve. The peroxidase method is not useful for the Solanum material studied.After standardized pollination the average number of haploid pollen grains on diploid stigmata was found to be 1625±127, that of diploid pollen on tetraploid stigmata 2863±98.A. W. B. Janssen 1975.     

Fertility and pollen tube growth in polyploid melons (Cucumis melo L.)

The high sterility of tetraploid melon (Cucumis melo L.) plants is a serious inconvenient to obtain economically useful polyploid lines. In order to study the alterations on diploid pollen tube growth and their influence on sterility, in vitro and in vivo pollen tube growth from three commercial varieties and its tetraploids was observed. Polyploid pollen grains show a lower germination percentage in vitro and a slower germination rate than haploid pollen. Furthermore, diploid pollen grains producing two or three tubes and tube branchings were observed on diploid pollen; this seems to indicate a decrease in its functionality. In vivo and in vitro pollen behaviours are quite similar, but an interaction between the pollen and the pistillar tissues was detected in vivo. In the diploid × tetraploid cross, where fruits were not obtained, pollen tubes showed a slow growth rate at the base of the pistil. The possible effects of these alterations in sterility are discussed in terms of the factors that could affect fecundation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Incompatibility in diploid and tetraploid crosses of Cucumis sativus and Cucumis metuliferus

The African horned cucumber (Cucumis metuliferus Naud.; 2x = 2n = 24) contains genes that can confer resistance to many important cucumber (C. sativus L.; 2x = 2n = 14) pests [e.g., root-knotnematode, Meloidogyne incognita (Kofoid & White) Chitwood]. Cucumber is highly susceptible to this root-knot nematode species, and a recent screening of C. sativus accessions in the U.S. National Plant Germplasm collection did not identify sources of resistance. Thus,autotetraploids of Cucumis sativus and C. metuliferus were created to recover fertile resistant interspecific progeny. Autotetraploids were obtained at the highest rate when seeds were immersed in 0.5% colchicine for a period of 6 to 8 hrs. Treatment durations less than 6 hrs produced few tetraploids, and durations of 10 hrs or more were lethal to seeds or developing seedlings. Crosses between C. sativus and C. metuliferus were made using diploid and tetraploid lines in all possible combinations, including reciprocals. Fruit development occurred in crosses when diploid and tetraploid C. sativus were used as the female parent. However, seeds developed only in fruit of C. sativus (4n) ×C. metuliferus (2n) crossings. Seeds from these crosses, however,were flat and not viable. No fruit development occurred in crosses whereC. metuliferus was used as the female parent. This revised version was published online in August 2006 with corrections to the Cover Date.     

Relationships between African species of the genus Cucumis L. Estimated by the production,vigour and fertility of F1 hybrids

Summary The production, vigour, and fertility of F₁ hybrids between nine African species of the genus Cucumis L. were studied as a measure of the relationships between the species. Hybrid plants were obtained from 29 out of the 72 possible cross combinations. Two F₁ hybrids died as seedlings, and 27 hybrids were raised to maturity. Pollen production and stainability varied greatly amongst these hybrids, as did fruit and seed set following self pollination and backcrossing with either parental species. The fruit shape of the hybrids was always intermediate between that of both parental species.Two species appeared to be closely related: C. prophetarum L. and C. anguria var. longipes A. Meeuse. Most other species produced highly to moderately fertile F₁ hybrids with at least one other species. C. metuliferus Naud. produced only sterile hybrids with C. zeyheri Sond. 2x. The results sustain the recent taxonomic classification of the genus (Jeffrey, 1980).     

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 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.     

Effects of aminoethoxyvinylglycine (AVG), environment,and genotype in overcoming hybridization barriers between Cucumis species

Summary It was attempted to overcome crossing barriers between the cucumber (Cucumis sativus L.), the melon (C. melo L.) and two wild Cucumis spp. (C. metuliferus Naud. and C. zeyheri 2x Sond.) by application of aminoethoxyvinylglycine (AVG) and by limiting the amount of rooting substrate. The reciprocal crosses between both wild species were used as a model system.Crosses between C. metuliferus and C. zeyheri 2x succeeded well if the maternal plants were grown in containers of 10 or 25 1 instead of in open soil. This treatment also improved crossability in the cross C. metuliferus x C. melo. It strongly enhanced the number of female flowers per plant in C. metuliferus. Application of AVG to pollinated flowers initially seemed also to improve crossability in these cross combinations, but later on this effect was not found again.Significant genetic variation for crossability was found in C. zeyheri 2x. Rare plants yielded numerous fruits with embryos after crosses with C. metuliferus and also with C. sativus. Clones from these plants gave similar results.Excessive temperature and radiation decreased crossability especially in crosses with C. metuliferus and C. sativus as maternal parent.     

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.     

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.     

Induction of 2n pollen in tulips by arresting the meiotic process with nitrous oxide gas

Triploid tulips have agronomically desirable traits such as vigorous growth and large flower size, but only a portion of all cultivated tulips is triploid. To apply 2n pollen to polyploid breeding of tulips, the polyploidizing agent, nitrous oxide gas (N₂O), was applied to bulbs. In tulips, meiosis in anthers occurs inside the bulbs from mid- to late-October. When meiosis in anthers (excised from bulbs) reached metaphase I, we treated other bulbs of the same clones with N₂O for 24–48 h. Most of the treated plants produced pollen grains with a wide-ranging or bimodal size distribution, indicating a mixture of n, 2n and aneuploid pollen grains. The use of pollen containing a relatively high proportion of giant pollen grains tended to yield larger numbers of triploids in the progeny. The number of giant pollen grains could be increased when N₂O-treated pollen grains were suspended in 10% sucrose and then sieved through a nylon mesh. Very few polyploids were observed in some cross combinations, even those involving pollen with a relatively high proportion of giant grains. Even so, this low polyploid yield most likely is due to a triploid block, because the capsules obtained in the crosses of the diploid×N₂O-treated plants contained some abnormal seeds, which were mostly triploid. Embryo culture was useful in rescuing abnormal embryos. The present study reveals that 2n pollen can be produced at high frequency using N₂O during tulip breeding.     

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.     

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.     

Meiotic chromosome behaviour in an allotriploid derived from an amphidiploid × diploid mating in Cucumis

A synthetic amphidiploid species Cucumis hyriviis Chen & Kirkbride (2n = 4x = 38; genome designated as HHCC) has recently been created from an interspecific mating between C. sativus L. (2n = 2x = 14: genome designated as CC) and C. hystrx. Chakr. (2n = 2x = 24; genome designated as HH). This amphidiploid is resistant to root knot nematode, tolerant to low irradiance, and has higher nutritional value than standard processing cucumber cultivars. An allotriploid (2n = 3x = 26; HCC) was derived from a cross between C. sativus L. and C. hytivus Chen & Kirkbride. Diploid meiotic behaviour in C. sativus and C. hystrix involves the development of seven and 12 metaphase bivalents respectively. In the derived allotriploid. univalents. bivalents, and trivalents (at relatively low frequency) were observed at metaphase I indicating that some homeologues from the C and H genomes can synapse. Based on a comparative karyotype analysis of cucumber (i.e. chromosome size and pairing behaviour) and aliotriploid plants, the seven bivalents observed at metaphase I were ascertained to be cucumber homologues, while the 12 univalents were of C. hystrix origin thus confirming the allotriploid karyotypic constitution to be HCC. On average, the frequency of trivalents was 0.24 at diakinesis and 0.22 in 100 meiocytes at metaphase I. indicating the possibility of genetic exchange due to the homoeology between the C and H genomes. After simultaneous cytokinesis, only polyads were observed in pollen mother cells (PMCs) at telophase II, which led to the production of sterile pollen grains. Multi‐polarization of chromosomes was dominant at anaphase II. However. in about 20% PMCs. chromosomes separated to form a 7C + 1‐2H complement, suggesting a possible method for the production of alien addition cucumber‐C hystrix lines through further backcrossing of the allotriploid to diploid cucumber.     

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.