Pollen germination and pollen tube growth following self-pollination and intra- and interspecific pollination of Medicago species

Summary Pollen germination and pollen tube growth after self- and intra- and interspecific pollinations of annual [M. scutellata (L.) Mill., M. disciformis DC., and M. rigidula (L.) All.] and perennial (M. sativa L.) Medicago species were studied using a Carl Zeiss microscope with a fluorescence filter attachment.Self-pollination of self-fertile annual and intraspecific pollination of cross-fertile perennials were characterized by high pollen germination and rapid normal pollen tube growth. In contrast pollen germination percentages were very low and many pollen tube growth abnormalities occurred following interspecific pollination and self-pollination of self-sterile plants. The time period from pollination to fertilization for interspecific crosses was about double that following self-pollination of the annual species. However, fertilization occurred frequently following interspecific pollinations. Much of the sterility was due to factor(s) operating after fertilization.Cooperative investigations of the Kansas Agricultural Experiment Station, Dept. of Agronomy and the U.S. Department of Agriculture, Agricultural Research Service. Contribution No. 82-582-J- Mention of a trademark name or proprietary product does not constitute a guarantee or warranty by the U.S. Department of Agriculture, and does not imply its approval over other products that also may be suitable.     

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 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.     

Cytogenetic evidence for the origin of teosinte (Zea mays ssp. Mexicana)

Summary Cytogenetic evidence has shown that teosinte (Zea mays ssp. mexicana (Schrad.) Iltes) and maize (Zea mays L. ssp. mays) are conspecific. They hybridize readily and their offspring are generally fertile. Teosinte could not have originated as a byproduct of maize-Tripsacum hybridization. Such introgression gave rise to plants that are phenotypically maize or Tripsacum, depending on which parent was used as a pollen donor. Compartive morphological and genetical studies indicated that it is more probable that maize originated from a teosinte-like ancestor under domestication, than that a maize-like plant gave rise to teosinte through a series of mutations.Reseach supported financially by the Illinois Agricultural Experiment Station, and Grants GB-40136-X and BM573-01034 A02 from the National Science Foundation.     

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.     

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.     

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.     

Barriers to interspecific hybridization in Ulmus americana

Summary Crossability barriers between the tetraploid Ulmus americana and five other diploid elm species were investigated using both fluorescence and scanning electron microscopy. Both pollen germination and pollen tube development were inhibited in all interspecific crosses. Pollen tube behavior on the stigma of U. americana was unchanged when pollen of a colchicine-induced U. pumila tetraploid was substituted for diploid U. pumila pollen.Self-incompatibility was observed in one of two American elms studied and manifested itself via reduced pollen germination and inhibited pollen tube growth on the stigma surface.The results indicate that crossing barrier(s) between U. americana and other elm species occur at the stigma surface and operate independently of ploidy level. This conclusion is discussed in relation to current breeding efforts to transfer Dutch elm disease resistance into the American elm via interspecific hybridization with Eurasian elms.     

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.     

Cytogenetic studies and the occurrence of triploidy in the wild potato species Solanum commersonii Dun.

Summary The South American wild potato species Solanum commersonii Dun. exists as two subspecies, subsp. commersonii and subsp. malmeanum (Bitter) Hawkes and Hjerting, and as both diploid and triploid cytotypes. The two subspecies were completely cross fertile but there were reciprocal differences in the number of seeds set per berry and the mean seed weight. Crosses between diploids and colchicine-induced autotetraploids yielded only a few seeds per berry, but 16 of the 18 germinated seeds were triploid or nearly so. Such a high proportion of triploids is unusual for such crosses in potatoes. Some triploids has a high percentage of stainable pollen. The origin of naturally occurring triploids is discussed in the context of 2n gametes, a triploid block, and endosperm balance numbers.     

Accelerating and synchronizing germination of strawberry seeds by osmotic pre-treatments

Summary Pre-soaking strawberry seed (Fragaria ananassa Duch.) in osmotic solutions accelerated and partially synchronized their germination. Pre-soaking advanced 50% germination from about 4 weeks to 4 or 5 days. Two to three weeks pre-soaking in a mineral solution of -10⁶Pa nominal osmotic potential at about 20 C was satisfactory. The resulting synchronization of germination could allow seedlings to be selected for rate of growth by directly comparing seedling in bulk sowings.     

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.     

Competition between 2x and x pollen in styles of Solanum tuberosum determined by a quick in vivo method

Summary Pollen competition in vivo was studied by placing two pollen samples on one stigma and comparing the length of the tubes under a U.V. microscope. Comparisons involving pollen of Solanum tuberosum and S. phureja showed that 2x pollen grew faster than x pollen, both in 2x and x styles. Consequences for induction of dihaploids in S. tuberosum are discussed.     

An S-allele survey of cabbage (Brassica oleracea var. Capitata)

Summary A total of 31 S-alleles was found in a survey of 197 cabbage plants representing 11 cultivars of diverse type. Most of these S-alleles also occurred in either kale or Brussels sprouts, but five of them have not been found previously and apparently occur only in cabbage. A more detailed study of five cultivars of spring cabbage showed only 12 S-alleles in all, with 6–10 S-alleles in four older cultivars and only 3 S-alleles in the newer more highly selected cultivar. S2 was by far the commonest S-allele, as it is in B. oleracea as a whole. The highly recessive alleles S5 and S15 were not particularly common in cabbage and this may partly explain why the sib problem in F₁ hybrids is apparently less in cabbage than in Brussels sprouts. Three cases were found in which an S-allele was completely recessive in both the stigma and the pollen. The problems for the breeder created by this rather unusual situation are discussed.     

Cytoplasmic homology between Aegilops mutica Boiss. and Ae. ovata L.

Summary Triticum aestivum L. em Thell. (2n=42; AABBDD), and T. durum Desf. (2n=28; AABB) genomes were substituted into the cytoplasms of Aegilops mutica Boiss. (2n=14; M_tM_t), Ae. heldreichii Holzm. (2n=14; MM), Ae. uniaristata Vis. (2n=14; M^uM^u), and Ae. ovata L. (2n=28; C^uC^uM^oM^o), to identify the M-genome diploid cytoplasm donor of Ae. ovata. Substitution of the T. durum genome into Ae. uniaristata cytoplasm resulted in a large proportion of shriveled inviable seeds. A few plump viable seeds were obtained all of which produced male-sterile plants having one univalent or telocentric chromosome from Ae. uniaristata. The T. aestivum plants having Ae. uniaristata or Ae. mutica cytoplasms were fertile. However, Ae. mutica was similar to Ae. ovata in the induction of delayed maturity and tall robust growth habit to the T. durum and T. aestivum plants. Cytoplasms of the other C- and M-genome diploids Ae. umbellulata Zhuk. (2n=14; C^uC^u) and, Ae. heldreichii (2n=14; MM) earlier had been shown to differ from that of Ae. ovata. Therefore, Ae. mutica is the most likely cytoplasm and M-genome donor to Ae. ovata.     

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.     

Slow-growth conservation of potato microplants: efficacy of ancymidol for long-term storage in vitro

Ancymidol was investigated as an alternative mediumsupplement to mannitol for slow-growth conservation ofpotato microplants in vitro. Differentconcentrations of ancymidol (0, 5, 10, 15, 20, 25, 30,35 and 40 M) were tested in slow-growthmedia based on MS medium supplemented with either 30or 60 gl^-1 sucrose. The cultures were conservedunder a 16-h photoperiod at two temperature regimesi.e. 24 ± 1 ^°C and 6 ± 1 ^°C. Therewere significant interactions between ancymidol andother factors such as sucrose, temperature andgenotype for microplant survival, microshoot heightand overall microplant growth. Ancymidol did have abeneficial effect on culture viability after prolongedmaintenance in vitro. The growth-inhibitingeffect of ancymidol persisted through a 16-monthculture period. Combined effect of ancymidol, sucroseand temperature showed that optimum culture viabilityand desirable microplant growth were obtained when thecultures were grown in MS medium supplemented with 10M ancymidol plus 60 gl^-1 sucrose at6 ± 1 ^°C. Vitrification and flaccidity, whichare very frequently observed in potato microplantcultures during prolonged maintenance in vitrounder osmotic stress (mannitol), were not observedwhen the microplants were conserved in ancymidolmedia. Genetic stability of potato microplantsconserved in ancymidol media was evaluated usingrandomly amplified polymorphic DNA (RAPD)fingerprints. Ancymidol did not induce any detectablegenetic variation in genomic DNA as visualized by theabsence of either any additional RAPD fragment oralterations in RAPD fragment patterns.     

Cytology and leafspot resistance in Arachis hypogaea x wild species hybrids

Summary Introgression of germplasm from diploid wild Arachis species to A. hypogaea has great potential for improving pest resistance in cultivated peanuts. This investigation evaluated methods for incorporating exotic germplasm into cultivated peanuts, especially for Cercospora arachidicola Hori resistance. Interspecific hybrids between A. hypogaea (cvs. NC 2 and NC 5) and the wild species A. cardenasii Krap. et Greg. nom. nud. and A. chacoense Krap. et Greg. nom. nud. were analyzed cytologically and for leafspot resistance. All F₁ hybrids were sterile, had irregular meiosis, and very few multivalents. They were highly resistant to C. arachidicola in field tests and had a 10-fold reduction of conidia per lesion in the greenhouse as compared to A. hypogaea cultivars. After colchicine treatments of F₁ hybrids, hexaploids (2n=60) and aneuploids (2n=54, 56, 63) were observed. The hexaploids had up to 18 univalents per pollen mother cell and very few multivalents, indicating a low frequency of intergenomic chromosome pairing. For C. arachidicola resistance, significant differences were not found among wild species parents, F₁ hybrids and two generations of hexaploids. Most hexaploids were stable at 2n=60 and embryos aborted when backcrosses with the respective wild species were attempted. However, when hexaploids were backcrossed to A. hypogaea, several fertile pentaploid (2n=50) offspring were obtained. Use of self-pollinating pentaploids is believed to be the quickest method to recover 40-chromosome hybrid derivatives in these hybrids.     

Allotetraploid hybrids produced by protoplast fusion for seedless triploid <Emphasis Type="BoldItalic">Citrus</Emphasis> breeding

In connection with crop improvement strategies for marginal regions it has been proposed to increase the outcrossing rate in barley which would presuppose high and persistent pollen viability to ensure successful cross fertilisation. The present study was designed to investigate the viability of mature pollen from extruded and non-extruded anthers of cultivated and wild barley (Hordeum vulgare ssp vulgare and ssp spontaneum, respectively) in comparison with the closely related but obligatory outcrossing species H. bulbosum. Pollen viability (PV) was assessed employing the p-phenylenediamine-peroxidase-test on pollen derived from spikes or anthers immediately after collection and after treatment at 20, 30 and 40 ^∘C for 1, 2, 4, 8 and 16 h. The latter treatment was interposed after 8 h by a 10 h period of darkness at 12 ^∘C and thus called 26 h treatment, consecutively. Initial PV was high with 98% across all genotypes and even at 40 ^∘C did not fall below 80% after 8 h. After the 26 h treatment, PV of two H. vulgare genotypes originating from semi-arid regions and of H. bulbosum fell below 60% while the other genotypes retained a PV of > 80%. Viability of pollen of extruded anthers in H. vulgare ssp. spontaneum was on average slightly but significantly lower than PV of non-extruded anthers but still remained above 90%, even after the 26 h treatment at 40 ^∘C. Pollen viability of the outcrossing species H. bulbosum ranged on a very similar level as PV of H. vulgare. Results indicate that pollen of H. vulgare retains a sufficiently high level of viability to ensure successful cross-fertilisation over a period of at least 26 h even at high temperatures of up to 40 ^∘C.     

Optimum plot size determination and its application to cucumber yield trials

Summary Methods of estimating Smith's b and, thereby, optimum plot size are compared from a theoretical viewpoint. For estimating b, generalized least squares is recommended over Smith's (1938) original method and other methods because the points used to fit the required regression are correlated and have unequal variances.Optimum plot size for once-over-harvest trials measuring yield (as number of fruits per plot) of pickling and frest-market cucumbers (Cucumis sativus L.) was estimated to be 0.7 to 3.8 m² (0.5 to 2.5 m of row for rows 1.5 m apart) for conventional harvesting, and 1.0 to 5.6 m² (0.7 to 3.7 m of row) for simulated harvesting using paraquat to defoliate plots before evaluation. Estimates of optimum plot size were calculated from a number of uniformity trials differing in year (1982 or 1983), planting date (early or late), and field. The estimates were sufficiently stable to suggest that they have useful generality.For multiple-harvest yield trials, optimum plot sizes for determining yield of pickling (expressed in $/ha or q/ha) or fresh-market cucumbers (i.e. USDA Fancy and No. 1 grade fruit combined or USDA Fancy, No. 1, and No. 2 grade fruit combined, in q/ha) were estimated from experimental data to be 6.4 to 10.3 m² (4.3 to 6.8 m of row).