Somatic embryogenesis in Vicia faba L.

The paper describes a method of somatic embryo induction in callus and suspension cultures of Vicia faba L. Callus was induced from immature cotyledons (green maturity stage) of white-flowering horse bean lines cultured on L2 medium (Phillips and Collins 1979) supplemented with 1% sucrose, 0.7% agar and different concentrations of 2,4-dichlorophenoxyacetic acid. The medium with 2.5 M 2,4-Dichlorophenoxyacetic acid was found optimum for embryogenic callus induction. Somatic embryos developed after transfer of the callus to media lower or zero 2,4-Dichlorophenoxyacetic acid and increased level of sucrose (2.5%). The release of somatic embryos from the callus was more apparent after transfer to liquid medium. There were various stages of somatic embryo development, i.e. globular, heart-shaped and torpedo ones.     

Sensitivity to drying in vitro of enzymes in mitochondrial subfractions from Vicia faba seed

In order to investigate the persistence of membrane and matrix functions following desiccation, enzymic activities were studied in Vicia faba L. seed mitochondrial subfractions subjected to drying and rehydration in vitro. Mitochondria were prepared after 0, 12 and 24 h of seed imbibition. These were fractionated into inner membranes (submitochondrial particles), outer membranes (12 and 24 h only) and the soluble matrix. Enzyme activities associated with the inner membrane and matrix were found to increase several-fold during the first 12 h of imbibition. The two matrix enzymes examined, malate dehydrogenase and glutamate dehydrogenase, were insensitive to in vitro drying at all stages of imbibition. The membrane-bound activities from 12 h and 24 h imbibed material, antimycin A-sensitive NADH: cytochrome c oxidoreductase and (F_o-F₁)-ATPase of the inner membrane and antimycin A-insensitive NADH: cytochrome c oxidoreductase of the outer membrane, were moderately sensitive to dehydration. The F₁-ATPase solubilized from the inner membrane (F_o-F₁) complex was much less sensitive to drying, provided this was done at room temperature.
Mitochondria posessing their outer membranes could not be prepared from dry seed. The antimycin A-sensitive NADH: cytochrome c oxidoreductase from inner mitochondrial membranes of unimbibed seed was extremely sensitive to desiccation in vitro, about 75 to 80% of the activity being lost. This loss could be somewhat reduced by addition of glycerol or sucrose before drying.
It is concluded that uncontrolled desiccation results in major damage to some of the membrane-bound enzymic systems in mitochondria, whereas activities in the soluble fraction are remarkably tolerant of desiccation.     

Inhibition studies on the interaction of Vicia faba lectin with carbohydrates.

Mono- and oligosaccharides and modified sugars have been studied quantitatively for their capacity to inhibit the agglutination reaction between Vicia faba lectin and yeast cells. The results seem to parallel the specificity of carbohydrate binding reported for concanavalin A.     

Isozyme gene markers in Vicia faba L.

Summary This study was conducted to assess the genetic basis of the variability observed for the glutamate oxaloacetate transaminase (GOT), Superoxide dismutase (SOD), esterase (EST), and malate dehydrogenase (MDH) isozyme systems in different open-pollinated Vicia faba varieties. Individual plants showing contrasting zymogram patterns were simultaneously selfed and cross-combined. Crossing was unsuccessful in producing progeny, and only selfed progenies were suitable for genetical analysis of isozyme variability. Three zones of GOT activity were made visible. The isozyme of GOT-2 and GOT-3 zones were dimeric and under the control of three alleles at the Got-2 locus and two alleles at the Got-3 locus, respectively. The isozymes of the GOT-1 zone did not show any variability. Three zones of SOD isozyme activity were made visible. The isozymes occurring in the SOD-1 (chloroplastic isozyme form) and SOD-2 (cytosol isozyme form) zones were dimeric and under the control of two alleles at the Sod-1 and Sod-2 loci. The isozyme visualized in the SOD-3 zone (mitochondrial isozyme form) were tetrameric and under the control of two alleles at the Sod-3 locus. Apparently the isozymes made visible in the most anodal esterase zones EST-1, EST-2, and EST-3 were monomeric, and the occurrence of two alleles at each of two different loci explained the variability observed in the EST-2 and EST-3 zones. For MDH, only two five-banded zymogram pattern types were found, and every selfed progeny showed only one of the two zymogram type, indicating that each individual possessed fixed alleles at the loci controlling MDH isozyme. Got-2, Got-3, Sod-1, Sod-2, and Sod-3 appear to be five new isozyme gene markers that can be useful in Vicia faba breeding for linkage study, varietal fingerprinting, outcrossing rate estimate, and indirect selection for quantitative characters.     

Cytoplasmic male sterility in Vicia faba L.

Summary In many higher plants, nucleo-cytoplasmic interactions lead to pollen abortion. In Vicia faba, cytoplasmic male sterility is unstable as the cytoplasm appears to shift from a sterile to a fertile state. In this report, five flower phenotypes are defined but the study is focussed on the progenies obtained from intermediate, semi-sterile plants with the same homozygous nuclear constitution during five successive generations. The results could be interpreted by quantitative modifications of at least four different kinds of cytoplasmic determinants.     

Intraspecific unilateral incompatibility in Vicia faba L.

Summary Unilateral incompatibility was discovered when crossing was attempted between different self-compatible types and subspecies of V. faba. Crossing in the direction female less self-fertile x male more self-fertile failed, whereas the reciprocal-crossing succeeded. Unilateral incompatibility developed with the evolution of less fertile and large seeded field bean types. How a cross fertilized (and self-incompatible) system may develop from a self fertilized one is discussed.The unilateral incompatibility in V. faba and other plant species is compared. The two-power competition hypothesis can explain all kinds of unilateral incompatibility reported so far in the literature. Breeding field beans for improved self-fertility is discussed.     

Agrobacterium-mediated transformation of Vicia faba

Among the major grain legume crops, Vicia faba belongs to those where the production of transgenic plants has not yet convincingly been reported. We have produced stably transformed lines of faba bean with an Agrobacterium tumefaciens-mediated gene transfer system. Stem segments from aseptically germinated seedlings were inoculated with A. tumefaciens strains EHA101 or EHA105, carrying binary vectors conferring (1) uidA, (2) a mutant lysC gene, coding for a bacterial aspartate kinase insensitive to feedback control by threonine, and (3) the coding sequence for a methionine-rich sunflower 2S-albumin, each in combination with nptII as selectable marker. Kanamycin-resistant calluses were obtained on callus initiation medium at a frequency of 10–30%. Shoot regeneration was achieved on thidiazuron containing medium in a second culture step. A subsequent transfer of shoots to BA-containing medium was necessary for stem elongation and leaf development. Shoots were rooted or grafted onto young seedlings in vitro and mature plants were recovered. Molecular analysis confirmed the integration of the transgenes into the plant genome. Inheritance and expression of the foreign genes was demonstrated by Southern blot, PCR, western analysis and enzyme activity assays. Although at present the system is time-consuming and of relatively low efficiency, it represents a feasible approach for the production of genetically engineered faba beans.     

Nitrogen Metabolism of Vicia faba

Vicia faba plants were grown for four and six weeks without externally supplied nitrogen. Some nitrogen was transported to the plant axis from the cotyledons throughout this period, but the amount available was insufficient to support maximum shoot growth. During this period the protein content of the shoot declined whilst the free amino acids, especially aspartic acid, glutamic acid, histamine and the combined pool for threonine, serine, asparagine and glutamine and ammonia, increased in amount. In contrast to the shoot the protein content of the root increased as did their free amino acid content, but the increase in the latter was less than in the shoot and only the combined value for threonine, serine, asparagines and glutamine increased significantly. During tbe last two weeks growth, some soluble non-amino acid compound appeared to donate nitrogen to the pool of free amino acids in the root and shoot.     

Pollen-Stigma Interaction in the Leguminosae: Stigma Organization and the Breeding System in Vicia faba L.

As in other papilionoid Leguminosae, the receptive surface ofthe stigma of Vicia faba L. is invested by a detached cuticle.This cuticle, the so-called ‘stigma membrane’ ofplant breeding literature, is lifted away from the epidermisduring development by the accumulation of a lipid-rich secretionreleased into the intercellular spaces of the stigma head bythe epidermal cells and the underlying three to four cell layers.The cuticle is thickened over the prominences left by the epidermalpapillae, thinning out between. Pollen, whether self or cross,cannot hydrate and germinate in contact with the intact stigmasurface, but must await the disruption of the cuticle and therelease of the retained secretion. In most genotypes this takesplace only when the flower is tripped by visiting pollinatorsor in consequence of severe agitation by wind. A comparison of lines differing in their degree of autofertilityin field conditions has revealed various differences in stigmastructure. A highly autofertile line had low papillae at thereceptive tip, with a relatively thin intervening cuticle, whilean autosterile line had longer papillae and a thicker cuticle.A line with partial autofertility was intermediate in thesecharacteristics. These properties of the stigma surface, togetherwith other differences in flower structure, are probably adequateto account for the variation in the degree of autosterility,since no evidence was obtained suggesting the presence of aneffective physiological self-incompatibility system in any ofthe lines studied. Since the rupturing of the stigma cuticle is affected by theturgor pressure of the cells of the stigma and style, some degreeof environmental interaction is to be expected: autofertilityshould be at the highest in conditions of adequate water supply,and lowest where there is water deprivation. Pollen-stigma interaction, Vicia faba L., Leguminosae, breeding system, self-incompatibility     

On the evolution of Vicia faba L.

Summary Diallel crosses between seven inbred lines obtained from different botanical groups including the two known subspecies of V. faba permitted study of the genetics of seventeen quantitative characteristics; there were very few significant reciprocal differences, and in all cases but one they were only weakly dependent on genotype. These results accord very well with those obtained from a study on population distances among thirty unselected populations of V. faba; many of these lie very close, forming a strong nucleus, which carries the maximum of potentialities of the species and from which different populations branch. Nevertheless differentiation occurs: the genetic system which regulates seed length shows overdominance in the positive sense in some lines, but partial or complete dominance in the negative sense in the rest of the studied lines. Interpretations suggesting that V. faba has suffered very little intraspecific differentiation are substantiated by the studies showing the presence of a partial incompatibility system; this is stronger in the Central European populations studied, weak (to various degrees) in the Spanish ones and absent in at least one population of the paucijuga group.     

Biosynthetic studies on chlorinated indoles in Vicia faba

We identified L-4-chlorotriptophan (L-4-Cl-Trp) in two week old plants of Vicia faba. Deuterium labeled indole was incorporated to 4-chloroindole in these plants. Deuterium labeled L- and D-4-Cl-Trp were both synthesized and, when supplied to the plants only L-4-Cl-Trp was incorporated to 4-Cl-indoleacetic acid, although the incorporation ratio was very low.     

Sister chromatid exchanges in Vicia faba

Evolutionary loss of the Y chromosome has occurred in Climacia areolaris (Hagen) of the neuropteran family Sisyridae. The diploid set comprises 6 pairs of autosomes, plus 2 X chromosomes in the female and 1 X in the male. The Y is retained in Sisyra vicaria (Walker) of the same family: its chromosome number is 14 in both sexes including 2X chromosomes in the female and 1X plus Y in the male. Two alternative pathways for the segregation of the sex chromosomes-distance segregation and sex bivalent formation-co-exist in the latter species in a ratio of approximately 1 to 6; the possible phylogenetic significance of this feature is discussed.     

Compartmentation in Vicia faba Leaves: I. Kinetics of C in the Tissues following Pulse Labeling

Leaflets of Vicia faba were pulse-labeled with ¹⁴CO₂ to follow the subsequent movement of photosynthate between leaf tissues. Samples were taken during a ¹²CO₂ chase, quick frozen, freeze-substituted, and embedded in methacrylate. Paradermal sections provided tissue samples consisting only of upper epidermis, palisade parenchyma, spongy parenchyma and veins, spongy parenchyma, or lower epidermis. Most CO₂ fixation occurred in the palisade parenchyma, but its ¹⁴C content declined rapidly after labeling. Concomitant with the decline of activity in the palisade parenchyma, there was an increase in activity in the spongy parenchyma and upper epidermis and a slow increase in the lower epidermis. Activity in the palisade parenchyma and spongy parenchyma eventually reached similar levels and remained constant. Tissue samples containing veins were consistently the most radioactive, and activity in those samples showed a decline. Very little change occurred in the insoluble fraction from any tissue. The results support previous assumptions regarding the pathway of assimilate transport to the veins, and demonstrate the rapidity of such transport. Sucrose is apparently the principal mobile compound.