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.     

Wilting of Leaves in Vicia faba L.

Wilting of leaves of Vicia faba L., which occurs when the pressurepotential (_p) is zero, and the leaf-water potential () at wiltingboth depend entirely upon the solute potential at incipientplasmolysis (_so) and not on soil-water status. Wilting in V.faba is acropetal; this is consistent with the hypothesis thatthere is a gradient of decreasing _so up the plant and that wateris transferred from the lower to the upper leaves, hasteningthe overall water loss from the lower leaves to the point when_p is zero. The gradient in _so up the plant is of the order of3–8 bar. It is proposed that wilting when _p>0 (i.e. > _so) shouldbe ‘apparent wilting’ and that when _p0 (i.e. _so),‘true wilting’.     

New Karyotypes of Vicia faba L.

Five new karyotypes of V. faba are presented and the types and positions of the structural changes combined in the construction of these new karyotypes are described. All their chromosomes are easily distinguishable by morphological criteria. These new chromosome complements are presently used to study the inter- and intrachromosomal distribution of induced chromatid aberrations and related problems.     

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.     

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.     

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.     

Lateral root formation in Vicia faba L.

MI was found to decrease in LP with increase in cell number, and reached minimal values just before the emergence of LP to form lateral roots. These changes in MI have been correlated with the accumulation of cells in G1, 24 hours before a lateral root is formed. The durations of C and the various phases of the mitotic cycle were also investigated in LP, and compared with those in small primordia. As lateral root primordia increase in cell number, the durations of C, S and G2 become longer, while G1 becomes shorter. Also MI and GF decrease while the proportion of quiescent cells increases. Thus, there is a gradual decrease in the rate of cell proliferation as primordia increase in size. The changes which take place in these parameters during lateral root primordium development have been compared with the events which occur in seed proliferating tissues at the onset of dormancy.     

Apical Dominance in Vicia faba

Apical dominance phenomena have been studied in seedlings ofVicia faba particularly in relation to the movement about theplant of uracil-2-¹⁴C applied to the cotyledons. Decapitationjust below the second node releases the growth of the lowermostlateral bud and inhibition is completely reimposed by applicationof indole-3-acetic acid (IAA) to the cut surface. Uracil-2-¹⁴Capplied in solution to the cotyledons is distributed in thestems of all experimental seedlings with no consistent differencesdue to decapitation or IAA application. On the other hand, decapitationresults in a rapid increase in uracil-2-¹⁴C content in the lateralbuds which far exceeds their promoted growth. This uptake iscompletely suppressed by IAA application. A ring of tri-iodobenzoicacid (TIBA)-lanolin paste around the stem above the bud suppressesIAA action both in bud growth and on uracil-2-¹⁴C uptake, andalso on the movement of IAA-1-¹⁴C down the stem. TIBA-application to the base of the bud does not prevent IAAaction on bud growth, but also does not prevent the movementof IAA-1-¹⁴C (or a water soluble product of its metabolism)into the bud. Direct application of kinetin to the lateral bud of intact plantscauses a short-lived release of growth. Gibberellic acid producesa smaller and scarcely significant increase which is additiveto the kinetin effect. Neither has any effect on uracil-2-¹⁴Cmovement into the bud. The implications of these findings are discussed in relationto various existing theories of the mode of auxin action inapical dominance and it is concluded that their strongest supportis for a mechanism involving the suppression of phloem differentiationin the vascular supply to the bud.     

Studies on Deoxyribonucleotide Synthesis in Vicia faba

The incorporation of uniformly labelled [¹⁴C] cytidine into the nucleic acids was studied in root tips of Vicia faba. Cytidine was found to be incorporated into RNA and DNA and the specific activities of the individual mononucleotides were deter- mined. The pyrimidine nucleotides were degrade and the ratio between the specific activity of the pentose and the specific activity of the base was determined for each nucleotide. CMP of RNA and deoxy CMP of DNA bad almost the same pentose: base ratios as The cytidine added to the incubation medium. It was concluded that the administered cytidine or a derivative of it was reduced to the corresponding deoxycytidine compound without breakage of the bond between pentose and base. [¹⁴C)-cytidine was transformed to UMP of RNA with some loss of radioactivity from the pentose and had almost the same pentose: base ratio as deoxy TMP of DNA. This indicates that the formation of thymidine phosphates involved The reduction of a uridine compound. Furthermore the incorporation of ¹⁴C-labelled thymidine, deoxyadenosine and deoxyguanosine into DNA was studied. Deoxyguanosine was found to be incorporated only to a slight extent. This finding has been discussed in relation to previous results.     

Environmental Control of Flowering in Vicia faba L.

There is a heteroblastic change in leaflet number in many stocksof Vicia faba, the rate of change being affected by the temperatureand photoperiod under which the plants are grown. In all exceptthe earliest flowering stocks of broad beans, and particularlyat high temperatures, flower initiation shows a quantita-tivelong-day response. For full development of the initiated inflorescenceslong days are required. Flower initiation may be accelerated in all except the earliestflowering stocks of V.faba by brief exposures to low temperatures,particularly when the plants are grown in short days at hightemperatures. The response to low temperatures is more rapidat I0° C. than at 4° C. but eventually approaches saturationat both temperatures. More prolonged exposure to low temperaturesdelays flower initia-tion. The response to low temperaturesincreases with increasing plant age but can occur during embryodevelopment on the mother plant. At temperatures above 14° C, and particularly above 23°C, a reaction inhibitory to flower initiation occurs. This reactionis probably restricted to the diurnal dark periods but is operativeat all stages of the life cycle, including embryo development.Its inhibitory effects may be overcome by subsequent cold treatment,and when the low temperature processes have reached saturationsubsequent high temperatures are no longer inhibitory. Although nucleosides could accelerate flower initiation, purineand pyrimidene analogues did not, with one exception, reducethe response to low temperature treatment.     

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.     

Effects of ozone on meiotic chromosomes of Vicia faba.

All of the levels of ozone used in these experiments caused morphological damage to plants of Vicia faba L., but only the dose of 200 parts per hundred million for 4 h or 8 h caused chromosomal damage in the microsporocytes. Significant chromosomal damage appeared 24 h after fumigation in metaphase I and anaphase I - telophase I but no significant damage was found in anaphase II - telophase II. This observation suggests that chromosomes are more susceptible to ozone during early stages of meiosis than at later stages. Chromosomal damage was of two types: physiological, as suggested by chromosome stickiness and physical, as indicated by bridges, fragments, and micronuclei.     

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