Intraspecific Variation in Mean Endosperm Cell Cycle Time in Vicia faba (L.) and Interspecific Hybridization with Vicia narbonensis (L.)

The rate of early embryo-sac development was studied in seven faba bean (Vicia faba L.) cultivars grown in a controlled environment. The embryo-sac stage was determined from the number of endosperm nuclei per embryo-sac and the number of embryo cells during the first 12 days after pollination. Differences in early embryo-sac development were observed between the genotypes studied. In addition, five different V. faba×V. narbonensis crosses and reciprocals were made using genotypes with small differences in mean endosperm cell cycle time and genotypes with large differences. The percentage of success (pod set) in V. faba×V. narbonensis crosses ranged between 9 % and 59 % while in the reciprocals it ranged from 12 % up to 30 %. However, cytological studies showed that the high percentage of success (pod set) observed in the cross A-107 × A-202 was not associated with a higher percentage of interspecific hybrid embryos. The results indicate that genotypes of the two species with smaller differences in mean endosperm cell cycle time result neither in a higher percentage pod set nor in bigger hybrid embryos.     

Early Embryo-Sac Development and Intraspecific Variation in the Mean Endosperm Cell Cycle Time in Vicia narbonensis (L.)

The early endosperm development in seven Vicia narbonensis populations was studied under a 23/ 16 ° C day/night temperature regime. Double fertilization occurred in less than 24 h after pollination. Endosperm divisions were synchronous until the 8th division of the endosperm nuclei. Aberrant endosperm nuclei were first observed alter the 8th division and their number increased after the 10th division. Endosperm nuclei continued to divide until the 12th day after pollination. During the following days a decrease in the number of endosperm nuclei was observed together with a substantial increase in the number of aberrants. Endosperm nuclei finally disintegrated. Neither cellularization nor starch deposition was observed in the endosperm. A positive correlation (r 0.69) was recorded between early pod length and the number of endosperm nuclei found in the ovules within each pod. In the populations studied the number of endosperm nuclei increased at a steady but different rate. This was followed by embryo development as well. It was concluded that intraspecific variation in the mean endosperm cell cycle time exists in Vicia narbonensis and that populations A 174 and A 202 should be more compatible to Vicia faba ev. ‘Polycarpe’ than population A 201.     

An embryological study of a Pisum sativum x Vicia faba cross

Summary Flowers of Pisum sativum plants grown in the field and greenhouse were self-pollinated and pollinated with Vicia faba. Germination of pollen and pollen tube growth were slower in the Pisum x Vicia cross. About 2% fertile ovules in the field and 8% in the greenhouse were obtained from the Pisum x Vicia cross, compared to 26% and 48% for Pisum x Pisum. Development of the Pisum x Vicia embryo was normal in form, though much slower than the Pisum x Pisum embryo, and about the sixth day it began to collapse. The greatest difference between the two types of crosses was observed in the endosperm. While the Pisum x Pisum endosperm nuclei divided normally and endosperm developed throughout the ovule cavity and around the embryo, the Pisum x Vicia endosperm nuclei formed only scattered masses of densely stained nuclei which were not always in close proximity to the hybrid embryo. It was concluded that faillure of some ovules to develop following Pisum sativum x Vicia faba cross pollination was the result of slow germination of pollen and slow growth of the Vicia pollen tubes, with subsequent lack of fertilization. Collapse of fertilized ovules was associated with abnormal and limited development of the hybrid endosperm, possibly leading to lack of nourishment and eventual collapse of the hybrid embryo.     

Pollination,fertilization and seed development in winter stocks of faba beans (Vicia faba L.)

Summary Incidences of pollination and fertilization were determined in five stocks of winter bean (two populations, two inbred lines, one cultivar) in two years. In the second year, irrigation was included as an experimental treatment and the development of seeds was monitored. Large differences between years were found in the measures of pollination and fertilization, and irrigation was associated with a small but significant increase in the proportion of ovules which were fertilized within fertilized flowers, particularly in the ovules furthest from the stigma. This increase was clearly reflected in the corresponding indices of seed maturation in the irrigated plants. Irrigation was not associated with any change in the proportion of ovules which aborted. Small but statistically significant differences in all of the measures of pollination and fertilization were found among the stocks. It is suggested that the variation in the incidences of pollination and fertilization was largely associated with variation in the synchrony of anthesis within racemes. The proportion of ovules within pods which matured as seeds and the proportion which aborted both varied significantly among the five stocks. Abortion appeared to act independently of fertilization in affecting the index of seed maturation, so both mechanisms should be considered when breeding for improved filling of pods.     

Electrophoresis of the Seed Proteins of Vicia faba L. and Its Immediate Progenitors — A Reappraisal

Electrophoretic patterns of total seed protein analyzed by polyacrylamide gel electrophoresis under non-dissociating and dissociating conditions were found to have diagnostic value for the Vicia species. The close similarity in the seed protein profiles of the cultivated species and Vicia narbonensis narbonensis indicated that this last species can be considered as the immediate progenitor of V. faba. The study also indicated that V. serratifolia should be included as a subspecies in V. narbonensis. The broad bean, Vicia faba, is one of the most important leguminous pulses cultivated over a wide range (from Central Asia to Western Europe and from Ethiopia and Sudan to Scandinavia). Schultz -Motel (1972) was the first to identify remains of V. faba seeds from archeological sites of the Bronze Age, mainly from Central and Southern Europe. Recently, Kislev (1985) found charred seeds of horsebean (V. faba L.) from the 7th millennium B.C. Thus, horsebean should be included among the legumes grown by some early Neolithic people. Zohary and Hopf (1973) have suggested that the wild ancestors of V. faba are species belonging to section Faba. In particular, the French vetch V. narbonensis L., was the promosing candidate; most probably, because the area of distribution of V. narbonensis coincides with that of V. faba. On the other hand, Ieibe (1934), Ladizinsky (1975), and Abdall and Gunzel (1979) refuted the reports that V. narbonensis (and wild species from the section Faba) may be considered as immediate ancestors of the cultivated species of V. faba. These authors reported on the albumin profile of seed protein in Vicia. This study aims to reassess the relationships between V. faba and its taxonomically related species in terms of their storage protein profiles and present some information on the evolution of V. faba.     

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)     

Controls over pollination and ovule fertilization in Vicia faba

Summary Variation in the frequencies of pollination and ovule fertilization was generated among faba bean (Vicia faba L.) genotypes in experiments which compared self-and open-pollination, natural and extended photoperiods, presence and absence of honey bees and a range of plant populations.There was a near-1:1 relationship between the frequencies of pollination of stigmas and the fertilization of at least one ovule within the pistil. Pollination was the major barrier to fertilization of the pistil, and ovules, since no mechanism was evident in the style to prevent pistil fertilization. The frequency with which single ovules were fertilized in pollinated pistils fell as their distance from the stigma increased, and averaged 83.5% overall.The frequency of pollination was most influenced by the level of autofertility possessed by the genotype, and the activity of pollinators. Provision of honey bees to plants in caged plots showed that high levels of pollination could be achieved under open-pollinated conditions, but this was also influenced by autofertility. Increasing plant population in the field had only a small effect on pollination, but extending photoperiod caused the levels and timing of self-pollination in three genotypes to vary.Large differences were found among genotypes in their early stigma receptivity, that is, the frequency of stigmas found to be pollinated at the erect standard stage, prior to the flower opening and being visited by bees. The implications of early stigma receptivity for producing new cultivars were discussed.     

Rate and inheritance of cross-fertilization in faba bean (Vicia faba L.)

Summary To assess the feasibility for synthetic varieties of Vicia faba L., 108 faba bean genotypes were screened for cross-fertilization rate in five field experiments carried out in 1989 and 1990 at Wageningen.Significant genotypic differences were observed for outcrossing rate, which ranged from 1 to 55%. These percentages, however, are thought to be not high enough for a commercially feasible production of synthetic varieties.The inheritance of cross-fertilization rate, determined by testing the F₄ generation of a cross between two inbreds, both with high outcrossing rates, appeared to be of a polygenic nature, as was indicated by the wide variation in, and the continous distribution of, the cross-fertilization rates. Among the F₄ lines tested some had a significantly higher cross-fertilization rate than the parents. However, this difference was not found when these lines were retested, indicating that possibilities for successful selection for higher outcrossing rates might be limited.     

The identification of faba bean (Vicia faba L.) trisomics by translocation tester set

Summary Five subtelocentric primary trisomics of Vicia faba L. were isolated from crosses between asynaptic mutants and normal plants or translocation tester stocks. The extra chromosome of each trisomic was identified cytologically by critical multivalent association at metaphase I of meiosis.     

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.     

Differential response of Vicia faba L. genotypes to 2, 3, 5-triiodobenzoic acid (TIBA)

Summary Response to application of the growth regulator 2, 3, 5-triiodobenzoic acid (TIBA) was examined in the parents and F₁ progenies of a diallel cross of six inbred lines of Vicia faba. TIBA affected the genotypes differentially but in general gave shorter internodes, reduced apical dominance, increased pod set on the lower nodes and earlier seed maturity.     

Inheritance and linkage of a gene for testa-imposed seed dormancy in faba bean (Vicia faba L.)

Seed dormancy in the grain legumes may interfere with germination in some situations, yet is not well described. Dormancy in faba bean (Vicia faba L.) was investigated to determine the mode of inheritance and whether the gene or genes controlling this trait are linked to other genes. Seeds produced by 235 recombinant inbred lines at the F₆ generation were used for this study. The parents were the cultivar ‘Optica’ and 172, an Afghanistan landrace which possesses extended seed dormancy. Germination was scored on a seed sample from each recombinant inbred line. Segregation patterns indicated that the trait is monogenic. This gene, named doz, is linked to a gene controlling anthocyanin and proanthocyanidin synthesis, sp-b, with the genetic distance estimated to be about 25 cM. No associations were detected between seed dormancy and pod dehiscence, seed lustre or mean seed weight. The implications of the persistence of a gene for seed dormancy in the gene pool of V. faba for the introgression of traits from landraces and cultivars possessing dormancy and for the origins of domesticated V. faba are discussed.     

Pattern analysis and genotypic x environment interactions in faba bean (Vicia faba L.) populations

Summary Twenty-nine populations of faba bean (Vicia faba L. subsp. major, equina and minor) of mediterranean origin were evaluated for the genetic variability of 12 biomorphological characters at four locations in Apulia and Basîli cata regions (Italy) in 1983–84.A pattern analysis appoach, based on classification and ordination procedures is presented for to characterize differences among population in mean performance and response across environments. Informations about G × E interaction were obtained.Research work supported by CNR, Italy. Special grant I.P.R.A. Sub-project 1. Paper N. 1099.     

The sulphur-rich Brazil nut 2S albumin is specifically formed in transgenic seeds of the grain legume Vicia narbonensis

Summary Epicotyl explants were co-cultivated with Agrobacterium tumefaciens EHA101 to transfer a chimeric 2S albumin gene construct carried in the binary Ti plasmid vectors pGSGLUC1 or pGA472 into the grain legume Vicia narbonensis. This gene encoding the sulphur-rich Brazil nut albumin was under the control of either the CaMV 35S promoter which permits gene expression in all organs, or the Vicia faba legumin B4 promoter which elicits seed-specific gene expression. After callus formation and selection for kanamycin resistance, somatic embryos were induced which, in the case of transformation with the vector pGSGLUC1, were screened for GUS activity. Embryos that produced GUS were in addition analysed for 2S albumin formation. Selected transgenic embryos were cloned by multiple shoot regeneration. Rooted and fertile plants were obtained by grafting transgenic shoots on the appropriate seedlings. R₁ and R₂ generations were raised and analysed for GUS as well as 2S albumin gene expression.Expression of the 35S promoter/2S albumin gene fusion took place in all organs of the transgenic plants including the cotyledons of seeds, whereas seed-specific gene expression was found in transformants with the legumin promoter/2S albumin gene fusion. The 2S albumin accumulated in the 2S protein fraction of transgenic seeds and its primary translation product was processed into the 9 and 3 kDa polypeptide chains. The foreign protein was localised in the protein bodies of the grain legume. Analysis of the R₂ plants indicated Mendelian inheritance of the 2S albumin gene. In homozygous V. narbonensis plants the amounts of 2S albumin were twice that present in the corresponding heterozygous plants. Whereas only low level formation of the foreign protein was achieved if the gene was under the control of the 35S promoter, approximately 3.0% of the soluble seed protein was 2S albumin if seed-specific gene expression was directed by the legumin B4 promoter. Some of these transformants exhibited a three-fold increase in the methionine content of the salt-soluble protein fraction extracted from seeds.Abbreviations 35S cauliflower mosaic virus 35S protein gene - GUS -glucuronidase - NPTII neomycin phosphotransferase II - LeB4 Vicia faba legumin B4 gene - 2S albumin Brazil nut (Bertholletia excelsa) 2S albumin - ER endoplasmic reticulum - rER rough endoplasmic reticulum - HPLC high pressure liquid chromatography     

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.     

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.     

Histological Observations on Interspecific Crosses in the Genus Lupinus

The hybridization barriers of interspecific lupin crosses were determined by histological investigations. The cross Lupinus hartwegii×L. mutabilis (both of American origin) revealed embryos in the cotyledonary stage as well as cellular endosperm. In the reciprocal combination, L. mutabilis×L. harwegii, embryo development stopped at the end of the globular stage, and simultaneously the coenocytic endosperm did no differentiate into the cellular state. In the combination L. luteus (European origin) ×L. hartwegii, embryo as well as endosperm development were initiated. Embryo development ended at any early globular stage. Twelve days after pollination the coenocytic endosperm had almost degenerated. With L. hartwegii as the female, no pollen germination could be observed.     

Self-fertility and the distribution of seed yield in Vicia faba L.

Summary Five lines of field beans (Vicia faba L.) bred and selected for a high level of self-fertility have demonstrated heritable differences in distribution of seed yield on the reproductive nodes when grown under conditions of selfpollination. Fully self-fertile genotypes produce a large proportion of the seed yield on the lower nodes. The breeding and selection for both self-fertility and the distribution of seed yield is discussed.     

Mutagenesis of faba bean (Vicia faba L.) and the identification of five different genes controlling no nodulation,ineffective nodulation or supernodulation

Summary Mutagenesis ofVicia faba L. using ethyl methanesulfonate has yielded three mutants (f13, f73, f245) with ineffective nodulation (Nod⁺Fix^–), one mutant (f48) unable to nodulate (Nod^–) and one supernodulating mutant (f32) having 3 to 5 times more nodules than the control in the absence of combined nitrogen, and expressing nitrate tolerant nodulation (Nod⁺⁺, Nts). All of the mutant phenotypes were controlled by single and distinct recessive genes designatedsyml tosym5, respectively. Grafting experiments showed that the (Nod⁺Fix^–) and (Nod^–) phenotypes were both under root genotype control and that the (Nod⁺⁺, Nts) phenotype was under shoot genotype control.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of faba bean <Emphasis Type="Italic">(Vicia faba</Emphasis> L.) using embryo axes

A system for the production of transgenic faba bean by Agrobacterium-mediated transformation was developed. This system is based upon direct shoot organogenesis after transformation of meristematic cells derived from embryo axes. Explants were co-cultivated with A. tumefaciens strain EHA105/pGlsfa, which harbored a binary vector containing a gene encoding a sulphur rich sunflower albumin (SFA8) linked to the bar gene. Strain EHA 101/pAN109 carrying the binary plasmid containing the coding sequence of a mutant aspartate kinase gene (lysC) from E. coli in combination with neomycinphosphotransferase II gene (nptII) was used as well. The coding sequences of SFA8 and LysC genes were fused to seed specific promoters, either Vicia faba legumin B4 promoter (LeB4) or phaseolin promoter, respectively. Seven phosphinothricin (PPT) resistant clones from Mythos and Albatross cultivars were recovered. Integration, inheritance and expression of the transgenes were confirmed by Southern blot, PCR, enzyme activity assay and Western blot.