Interspecific hybrids of Cyclamen persicum Mill. and C. purpurascens Mill. produced by ovule culture

Summary Interspecific crosses were made to introduce the scent of flowers of C. purpurascens into C. persicum cultivars and ovule culture was used to rescue the abortive hybrid embryos. Cultivars of C. persicum diploid (CPD, 2n=2×=48) and C. persicum tetraploid (CPT, 2n=4×=96) were the pistillate parents and wild species of C. purpurascens (CP, 2n=34) were staminate parents. After pollination, crossed ovaries were collected periodically and examined using paraffin sections. Histological observations suggested that both hybrid ovules of CPD x CP and CPT x CP should be transferred to culture medium 35 days after pollination. Based up on this observation, crossed ovaries were collected 28 days after pollination and ovules with placenta were transferred to MS (1962) medium containing 3% sucrose. These ovules were cultured in the dark at 25° C. The hybrids (2n=41) derived from CPD x CP had the scent of C. purpurascens, whereas the hybrids (2n=65) derived from CPT x CP had the scent of C. persicum. Although both hybrids had complete genomes from the parents and produced a few viable pollen grains, they failed to yield viable seeds by self- and cross-pollination with fertile pollen grains of C. persicum cultivars.Abbreviations CPD C. persicum diploid - CPT C. persicum tetraploid - CP C. purpurascens     

Amphidiploids between Cyclamen persicum Mill. and C. purpurascens Mill. induced by treating ovules with colchicine in vitro and sesquidiploids between the amphidiploid and the parental species induced by conventional crosses

Summary We cultured colchicine-treated hybrid ovules in vitro to produce fertile amphidiploids of C. persicum (2n=2x=48. referred to as AA) × C. purpurascens (2n=2x=34, referred to as BB). Seedlings and mature plants were obtained from the ovules without colchicine and those exposed to 50 mg/l colchicine for 5, 10 and 15 days, whereas they were not obtained from the ovules exposed to 50 mg/l colchicine for 20 days and 500 mg/l for 5, 10, 15 and 20 days. Although 8 mature hybrids derived from the ovules without colchicine produced a few fertile pollen grains, they failed to produce viable seeds by self-fertilization. The hybrids had 41 somatic chromosomes. Four and 3 mature plants were derived from ovules exposed to 50 mg/l colchicine for 10 and 15 days, respectively. One each among 4 and 3 mature plants showed a high frequency of pollen grain fertility, produced several seeds by self-fertilization, and had 82 somatic chromosomes which is twice the number of hybrid chromosomes (2n=41, AB). These findings indicated that these plants are amphidiploids (2n=82, AABB) between C. persicum and C. purpurascens. Three and 2 viable seeds were derived by the conventional crosses of diploid C. persicum × the amphidiploid and the amphidiploid × C. purpurascens, respectively. Flowering plants that developed from the seeds of diploid C. persicum × the amphidiploid were barely fertile and had 65 somatic chromosomes (2n=65, AAB), whereas those that developed from the seeds of the amphidiploid × C. purpurascens were barely fertile and had 58 somatic chromosomes (2n=58, ABB). The somatic chromosomes indicated that these plants are probably sesquidiploids between the amphidiploid and either C. persicum or C. purpurascens. The interspecific cross-breeding of cyclamen using the amphidiploids and the sesquidiploids is 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.     

Hybridization between diploid (Gossypium arboreum) and tetraploid (Gossypium hirsutum) cotton through ovule culture

Summary With in vitro culture of ovules, interspecific hybrids have been obtained in an otherwise incompatible cross between a diploid (Gossypium arboreum) and a tetraploid (G. hirsutum) cultivated cotton. The early abortion of the embryo was prevented by repeated treatment of the flowers, immediately after pollination with a solution of gibberellic acid and naphthalene acetic acid. The ovules excised three days after pollination and cultured in a liquid medium underwent profuse proliferation, whereas on an agar-solidified medium supplemented with casein hydrolysate, indoleacetic acid and kinetin they germinated to form hybrid plants.     

Interspecific hybridization inBrassica juncea × Brassica hirta using embryo rescue

Summary Interspecific hybrids have been obtained in an otherwise incompatible cross betweenBrassica juncea × Brassica hirta through the in vitro culture of hybrid ovules and ovaries. The best response was observed from ovules and ovaries cultured 10–15 and 5–7 days after pollination respectively on a basal medium supplemented with indoleacetic acid, kinetin and casein hydrolysate. In some cases the basal cut end of the ovaries proliferated to form callus and shoots. The in vitro-derived hybrid seeds varied in their colour, size and shape, and the F₁ plants in the field showed a large diversity in their morphological traits. The hybrids were sterile, and had an intermediate number of chromosomes (2n=30).     

Production of interspecific hybrids between Alstroemeria pelegrina L. var. rosea and A. magenta Bayer by ovule culture

Interspecific hybrids were efficiently produced in the cross-incompatible combination between Alstroemeria pelegrina L. var. rosea and A. magenta Bayer by culturing immature ovules with placenta 7–14 days after pollination on 2 g/l Gelrite-solidified MS medium containing 3% (w/v)sucrose. The plants showed intermediate characteristics between the parents and their hybridity was confirmed by karyotype and DNA analyses. The mean number of chromosome association per PMC at metaphase I was 2.60I+6.70II, pollen stainability was20.8%, and they produced viable seeds after self-pollination. Furthermore, mature plants were obtained when the hybrids were backcrossed as male parents with both the parents. The backcross-progeny from A. pelegrina var. rosea × hybrids exhibited 3.8 to 79.7% pollen stainability and that from A. magenta × hybrids 78.8 to 98.3%. Almost all of these plants produced viable seeds after self-pollination, which implies that they can beutilized for breeding of novel cultivars of Alstroemeria. This revised version was published online in July 2006 with corrections to the Cover Date.     

Intersubgeneric crosses within the genus Pelargonium

Summary The two cultigens, Pelargonium X domesticum (n=11), subgenus Pelargium, and P. X hortorum (n=9), subgenus Ciconium, were crossed reciprocally and with P. peltatum (n=9), subgenus Dibrachya. Diploid and tetraploid cultivars of P. X hortorum and P. peltatum and tetraploid cultivars of P. X domesticum were used. Viable seeds were obtained from crossing P. X hortorum and P. peltatum and four hybrid plants were produced. Only fruit set occurred in the cross P. X domesticum x P. X hortorum. In all other combinations no fruit set and no seeds were produced.In P. X domesticum x P. X hortorum and P. X domesticum x P. peltatum pollen tubes penetrated the stigma but not the style or ovary. Although the styles elongated in P. X domesticum x P. X hortorum, the ovules did not grow. Fruit set also occurred when the emasculated florets of P. X domesticum were bagged and not pollinated. Fruit set in this cross was apparently a physiological response of the plant, perhaps to the cold temperature, 5°C, used to induce flowering and not a response to either pollination or fetilization.     

Interspecific hybridization between Vicia faba (L.) and Vicia narbonensis (L.): Early pod growth and embryo-sac development

Summary Fertilized embryo-sac development and pod growth was studied in one Vicia faba cultivar, one Vicia narbonensis population and their reciprocal crosses. The initial development of endosperm and embryo was at least four days faster in V. narbonensis than in V. faba. Pods and ovules developed also faster in V. narbonensis than in V. faba. The growth rate of the hybrid pods followed the growth rate of the mother species, but was slower than that of the pods from selfed flowers. In the cross V. narbonensis × V. faba the ovules stopped growing 9 days after pollination, while in the reciprocal cross they stopped growing 15 days after pollination. Hybrid embryo-sacs from V. faba × V. narbonensis were aborted before they reached the stage of 256 endosperm nuclei or 200 embryo cells. Selfed V. faba embryo-sacs reached this stage in less than 9 days after pollination. In the reciprocal cross the embryo-sacs were aborted before they reached the stage of 128 endosperm nuclei or 80 embryo cells. Selfed V. narbonensis embryo-sacs reached this stage at the 4th day after pollination. Given that at these stages the embryo has less than 200 cells it was concluded that an in-ovule embryo culture technique should be developed to obtain hybrid plants.     

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.     

The transfer of triazine resistance from Brassica napus L. to B. oleracea L. III. First backcross to parental species

Summary Atrazine resistant Brassica napus × B. oleracea F₁ hybrids were backcrossed to both parental species. The backcrosses to B. napus produced seeds in both directions but results were much better when the F₁ hybrid was the pollen parent. Backcrosses to B. oleracea failed completely but BC₁s were rescued by embryo culture both from a tetraploid hybrid (2n = 4x = 37; A₁C₁CC) and sesquidiploid hybrids (2n = 3x = 8; A₁C₁C). Progeny of crosses between the tetraploid hybrid and B. oleracea had between 25 and 28 chromosomes. That of crosses between the sesquidiploid hybrid and B. oleracea had between 21 and 27. A few plants that had chromosome counts outside the expected range may have originated from either diploid parthenogenesis, unreduced gametes or spontaneous chromosome doubling during in vitro culture. Pollen stainability of the BC₁s ranged from 0% to 91.5%. All the BC₁s to B. oleracea were resistant to atrazine.     

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.     

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.     

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.     

Influence of selected wheat and rye genotypes on the direct synthesis of hexaploid triticale

Summary In the synthesis of primary hexaploid triticale, a cross-incompatibility barrier exists when tetraploid wheat (4X) is crossed with diploid (2X) rye. Fertilization may occur, however, abnormal endosperm development usually leads to premature embryo death. Four selected tetraploid wheat lines were crossed as females with seven open-pollinated rye lines and the resulting embryos were rescued in vitro 13–16 days after pollination. The wheat genotypes showed a major influence on crossability (seed set), embryo development and plant recovery. The highest efficiency of amphihaploid plant recovery (18.3 plants per 100 pollinated florets) was obtained from one 4X wheat line originally selected from the cross T. carthlicum × T. dicoccoides. Some of the 3X amphihaploid plants (ABR) derived from two wheat lines showed relatively high level of partial fertility presumably as a result of meiotic restitution. Correlation analysis showed that crossability (seed set), normal hybrid embryo development in vivo and embryo culturability were independent of each other.     

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.     

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 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.     

Genotypic variation and polymorphism of 2S albumins of sunflower

Summary An early postfertilization ovule culture technique in Alstroemeria was described in which young ovules were cultured in vitro two days after pollination. This resulted in the direct production of seedlings in a normally incongruous cross combination. A histological study of the in vitro cultured ovules revealed the development of the embryo inside the ovule in the absence of normal endosperm. The in vivo grown ovules showed a total breakdown of the developing seed within four weeks after pollination due to a postfertilization barrier. This ovule culture technique was applied in a diallel cross of seven Alstroemeria species, five from Chile and two from Brazil. The selfings and the reciprocal crosses between the Brazilian species A. inodora and A. brasiliensis were congruous and gave seed set. The cross A. inodora x A. pelegrina gave viable seeds. In all other 39 combinations no viable seeds were harvested. In those cross combinations both pre-and postfertilization barriers were found. After early ovule culture hybrid plants were obtained in 27 of the 39 incongruous species combinations of the diallel.Abbreviations DAP days after pollination - MS medium medium according to Murashige & Skoog (1962)     

Fingerprinting of alfalfa meiotic mutants using RAPD markers

Summary A calendar of female sporogenesis and gametogenesis was made for both apomictic tetraploid (2n=4x=36) Brachiaria brizantha and Brachiaria decumbens and their apomictic F₁ hybrids with sexual tetraploid (2n=4x=36) Brachiaria ruziziensis. Microgametogenesis was used as a reference. Apospory was facultative in both species and hybrids. Environmental conditions had variable effects on the level of apomixis according to each genotype. Ratios of segregation into sexuals and apomicts in the interspecific hybrids suggest an oligogenic determinism with dominant apomixis in the genus Brachiaria. Highly apomictic and partially male fertile hybrids were identified and will be used in an improvement program to transfer genes for apomixis into the sexual species B. ruziziensis.     

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.