Stereological analysis of cotyledon tissue inVicia faba L. var. aquadulce during germination

Summary Stereological analysis was carried out on cotyledon tissue at three different stages of germination. The tissue was selected by reference to adjacent sections which were assayed histochemically for protease activity. Day six tissue had no activity while eight and 14-day tissue did. Results show that during germination both tissue and cellular components undergo changes. The cells increase in size, the intercellular spaces increase, the cytoplasm increases in volume, the protein bodies swell and fuse, and small starch grains appear while the large starch grains do not undergo any major changes.     

Steric hindrance and its effect on chromosome movement inVicia faba somatic cells

Summary The arrangement of chromosome arms in metaphases and anaphases has been studied inVicia faba root meristem cells. During metaphase, the long chromosome arms are aligned parallel to the spindle axis. As a consequence, at the onset of anaphase, one chromatid can move straight ahead to the spindle pole whereas the other has to invert its orientation. Specially in narrow cells it has been observed frequently that some chromatids move in a reverse orientation to the pole, i.e., they move telomere-first instead of centromere-first. This behaviour results in a chromatid which protrudes beyond the main group of late anaphase or telophase chromatids. It is dicussed that the most likely explanation for the phenomenon is that in narrow cells chromatid behaviour is influenced by steric hindrance by the tightly packed surrounding chromatids and microtubules. When there is insufficient room, some chromatids are unable to make the required U-turn. Under such conditions the kinetochore of a non-inverted chromatid pulls the chromatid in a reverse orientation to the pole. An alternative explanation, i.e., protruding chromatids being the result of a neocentric activity at the telomere end of a reverse-directed chromatid or the lateral associations of spindle microtubules, failed to find support by electron microscopical studies.     

Cytological localization of fast renaturing and satellite DNA sequences inVicia faba

Summary DNA sequences reassociating within a Cot value of 1.8×10^–1 and those producing a light satellite in a CsCl density gradient were isolated fromVicia faba DNA and hybridizedin situ on squashes of roots of the same species. Silver grains were seen to be scattered over both the interphase nuclei and the metaphase chromosomes after hybridization with fast renaturing DNA sequences, indicating these are fairly regularly interspersed in theV. faba genome. Clustered labeling occurred after hybridization with satellite DNA sequences, indicating these are clustered in the genome. The localization of satellite DNA in chromosomes appeared to correspond closely to the position of the bright bands detectable after staining with quinacrine mustard. After hybridization with both DNA probes, labeling intensity over the nuclei of meristematic cells was higher than that over the nuclei of differentiating and/or differentiated cells. These results are discussed in relation to the structure of the cell nucleus, the mechanism of quinacrine banding and to previous data suggesting underrepresentation of nuclear repeated DNA sequences in differentiatingV. faba root cells.     

Cell biological mechanism for triggering of ABA accumulation under water stress inVicia faba leaves

Water stress-induced ABA accumulation is a cellular signaling process from water stress perception to activation of genes encoding key enzymes of ABA biosynthesis, of which the water stress-signal perception by cells or triggering mechanism of the ABA accumulation is the center in the whole process of ABA related-stress signaling in plants. The cell biological mechanism for triggering of ABA accumulation under water stress was studied in leaves ofVicia faba. Mannitol at 890 mmol ·kg^-1 osmotic concentration induced an increase of more than 5 times in ABA concentration in detached leaf tissues, but the same concentration of mannitol only induced an increase of less than 40 % in ABA concentration in protoplasts. Like in detached leaf tissues, ABA concentration in isolated cells increased more than 10 times under the treatment of mannitol at 890 mmol · kg^-1 concentration, suggesting that the interaction between plasmalemma and cell wall was essential to triggering of the water stress-induced ABA accumulation. Neither Ca²⁺-chelating agent EGTA nor Ca²⁺channel activator A23187 nor the two cytoskeleton inhibitors, colchicine and cytochalasin B, had any effect on water stress-induced ABA accumulation. Interestingly water stress-induced ABA accumulation was effectively inhibited by a non-plasmalemma-permeable sulfhydryl-modifier PCMBS (p-chloromercuriphenyl-sulfonic acid), suggesting that plasmalemma protein(s) may be involved in the triggering of water stress-induced ABA accumulation, and the protein may contain sulfhydryl group at its function domain.     

Argyrophilic nucleolus-associated chromatin in epidermal cells ofVicia faba induced by cold treatment

Summary The effect of growth temperature on the structural organization of the nucleolus in the epidermal cells ofVicia faba was studied with the silver staining technique at the light and electron microscopic levels. In plants grown at 22 °C, the nucleoli of the epidermal cells were poorly developed, most were less than 3 m in diameter and they occasionally carried minute fluff-like or particle-like accessories. When the plants were transferred into 5 °C incubator, moderately silver-impregnated spherules (MIS) with diameters of about 1.0 to 1.5 m were discerned on the surfaces of the nucleoli. The incidence of nucleoli with the MIS rapidly increased within a few days and thereafter increased little by little up to 40 days at which time 90% of the nucleoli carried the MIS. The unfused nucleoli usually had a single MIS but most of the fused nucleoli had two MIS; in other words, most cells had two MIS per cell. On the other hand, when the plants grown for a prolonged time at 5 °C were transferred back into the 22 °C incubator, the proportion of nucleoli with the MIS drastically decreased within a day. Silver staining at the electron microscopic level revealed that the MIS exactly corresponded with the compact block of nucleolus-associated chromatin, since this compact chromatin block was significantly covered with silver grains while other chromatin was not. The present findings suggest that growth at low temperature allows incorporation of the argyrophilic nucleolar substance into the compact block of nucleolus-associated chromatin, resulting in the appearance of the MIS.     

Genetic changes under domestication in Vicia faba

Summary The components of variation within each one of two sets of landraces and/or cultivars of Vicia faba, respectively constituted of primitive and advanced morphological types, were studied by means of two sets of 8 × 8 diallel crosses with two repetitions. The results show that primitive and modern forms differ from each other in both the intensity and the kind of selective pressures acting on them, mainly on those characters more modified through the domestication process: i.e., seed morphology and the number of flowers per node. Because of the paramount importance of the additive component in the primitive forms, it is suggested that the most important type of selection on them is the stabilizing one. On the contrary, in the most advanced forms the selection is directional and disruptive : directional towards greater yields, and disruptive separating two morphological types, major and equina. The plant response to these different selective pressures has been to modify the genetic control of different characters: thus the primitive forms generally show only additivity while the most advanced forms show additivity as well as directional and asymmetrical dominance.     

Purification and characterization of trypsin inhibitor from seeds of faba bean (Vicia faba L.)

A trypsin inhibitor from seeds of faba bean (Vicia faba L.) was purified to near homogeneity as judged by native-PAGE with about 11 % recovery using ammonium sulphate fractionation, ion-exchange chromatography on DEAE-cellulose and gel filtration through Sephadex G-100. The inhibitor had a molecular weight of 18 kD as determined by SDS-PAGE and Sephadex G-100. The inhibitor inhibited trypsin and chymotrypsin to the extent of 48 and 12 %, respectively. The inhibtion was of non-competitive type with dissociation constant for the enzyme inhibitor complex in the region of 0.07 mg·ml⁻¹. The inhibtor was stable between pH 4 and 5. It completely lost its activity when heated at 125 °C for 1 h or at 100 °C for 2 h. The inhibitor also lost its activity on exposure to 2-mercaptoethanol. Based on these properties, it could be concluded that Vicia faba trypsin inhibitor belongs to Bowman-Birk type of inhibitors, as it has molecular weight lower than generally observed for Kunitz type inhibitors.     

Genetics and mapping of new isozyme loci in Vicia faba L using trisomics

Polymorphism in ten enzyme systems (ACO, ACP, AAT, EST, FK, ME, NAG, PRX, 6PGD, and SOD) in Vicia faba L. was analyzed, revealing 13 loci, six of which have not been reported before. Inheritance, genetics, possible location, and linkage analysis were studied in 13 different F² populations trisomic for four of the six chromosomes (nos. 3, 4, 5, and 6) of the species. Each of these loci exhibited typical Mendelian inheritance except for those involved in the trisomic chromosome. Five loci have been assigned to a specific chromosome: Est-2 to chromosome 3, Fk-2 to chromosome 4, Prx-1 to chromosome 5, and Sod-1 and Pgd-p to chromosome 6. Nag-1 and Pgd-c displayed a linkage of 22.8 cM indicating a clear homology with chromosome 5 of garden pea on which both markers are syntenic.     

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.     

Rhizobium leguminosarum inoculation decreases damage to faba bean (Vicia faba) caused by broad bean mottle bromovirus and bean yellow mosaic potyvirus

Faba bean (Vicia faba) plants were inoculated with rhizobia and then their sap was infected with broad bean mottle bromovirus (BBMV) or bean yellow mosaic potyvirus (BYMV) in a field experiment. Both viral infections significantly decreased shoot and root dry weight, number of nodules, nodule dry weight, numbers of flowers and pods/plant, total plant N, grain yield and N₂ fixation. However, inoculation withRhizobium leguminosarum significantly increased all these parameters, both in healthy and virus-infected plants. Although BYMV was more destructive than BBMV, inoculation with rhizobia could be used, with other control measures, to limit damage by both viruses.The authors are with the Department of Biochemistry and Soil Science, Faculty of Agriculture, Shambat, Sudan.     

Molecular heterogeneity and genetics of Vicia faba seed storage proteins

Summary Legumin and vicilin were purified from seeds of Vicia faba L. var. Scuro, characterized in different electrophoretic systems, and used to produce polyclonal antibodies in rabbits. Two-dimensional electrophoretic studies showed a wide range of heterogeneity in the subunits of both legumin and vicilin. Legumin was found to be composed of 29 disulphide-linked subunit pairs with different molecular weight and/or isoelectric point. Western blot analysis of legumin of several mutants revealed molecular polymorphism based on a corresponding gene family. Three different -major legumin patterns were found, and inheritance studies showed that the 34.3-kD legumin polypeptide is the product of one locus, Lg-1, which is the first legumin genetic locus described in Vicia faba. Vicilin was found to be composed of as many as 59 subunits distributed in a molecular weight range of 65.7 to 42.8 kD (major polypeptides) and 37.2 to 15.2 kD (minor polypeptides), with different isoelectric points. A model is proposed that explains the possible formation of the minor subunits and the major subunits of 48.2 and 46 kD molecular weight (MW) from proteolytic cleavages and/or glycosilation of precursor polypeptides. Ten different vicilin electrophoretic patterns were observed among the analyzed accessions, which showed large molecular polymorphism that proved to be under genetic control.Contribution no. 55 from the Center of Vegetable Breeding, Portici, Italy     

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.     

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.     

Nucleolus-like structures detected in the cytoplasm ofBrodiaea uniflora: Light- and electron-microscopic surveys during mitosis

Summary Both light- and electron-microscopic studies confirmed the presence of cytoplasmic nucleolus-like structures in the root-tip meristems ofBrodiacea uniflora, Liliaceae. This structures were found to be different from all the types of nucleolus-like structures previously detected in the cytoplasm. The nucleolus-like structures in the present material usually appeared as complex structures with two types of elements, although the two elements sometimes lay separately in the cytoplasm. The two elements were differentially stained by the silver staining technique: one reacted more intensely with silver forming black-stained dot while the other was stained brown. The ultrastructural examination revealed that the black-stained dots were spherical, about 1.0 m in diameter, and consisted of highly electron-dense material in which many lacunar areas were always seen, while the brown-stained spherules were consisted of loosely packed RNP- or ribosome-like granules. The nucleolus-like structures were found almost exclusively at telophase and they were never or seldom detected from interphase to anaphase.Their peculiar behavior during mitosis and their ultrastructural organization are discussed with reference to their origin.     

Interaction of nitrate uptake and nitrogen fixation in faba beans

An experiment was carried out to determine the relationship between nitrate uptake and nitrogen fixation of faba beans. Therefore inoculated and uninoculated faba beans were grown in nutrient solution with different nitrate concentrations. Nitrate uptake was measured every two days during the growing period. At the end of the experiment the nitrate uptake kinetics were determined with a short time depletion technique and nitrogen fixation was measured with the acetylene reduction method. A limitation of nitrate uptake due to nitrogen fixation was relatively small. Nitrate concentrations of approximately 1 mol m^–3 and 5 mol m^–3 decreased nitrogen fixation to values of 16% and 1% of the control plants which received no nitrate nitrogen. A reduction of nitrogen fixation was mainly due to a decrease of specific nitrogen fixation per unit nodule weight and to a lesser extent due to a reduction of nodule growth. Only the maximum nitrate influx (I_max) seemed to be influenced by nitrogen fixation. Michaelis-Menten constants (K_m) and minimum NO _inf3 ^– -concentrations (C_min) were not significantly influenced by nitrogen fixation.     

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.     

Studies on the movement and distribution of ethylene in Vicia faba L.

In Vicia faba ethylene does not appear to move between different parts of the plant in physiologically significant amounts. The resistance to longitudinal movement is such that lateral emanation effectively isolates different parts of the plant from each other. When emanation is prevented, ethylene can be channelled to any part of the plant. Exposure of one section of a plant to ¹⁴C-labelled ethylene (up to 200 l/l) increased the internal concentration in other parts with ethylene that did not originate from the feeding chamber. A basipetal gradient of endogenous ethylene concentration was found in the lacuna of intact plants, the source of ethylene being the stem tissue. The permeability of stem tissue to ethylene decreases with age. The concentration of ethylene in tissues surrounding the lacuna is always higher than that in the lacuna and it is argued that compartmentation of ethylene occurs within these tissues.     

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.