A combination of thidiazuron and benzyladenine promotes multiple shoot production from cotyledonary node explants of faba bean (Vicia faba L.)

A procedure for multiple shoot formation from cotyledonary node explants of faba bean (Vicia faba L.cv.S.Ghdar) cultured on MS medium containing benzyladenine (BA) and thidiazuron (TDZ) was developed. Explants on medium with TDZ in combination with BA produced a higher number of shoots than with either cytokinin alone. The highest number of shoots was obtained when explants from 7-day-old seedlings were cultured on MS medium supplemented with TDZ and BA (2 mgl–1 each) for 31 days before transfer to hormone-free MS medium for elongation. Shoots produced in vitro were rooted on half-strength agar-solidified MS basal medium or with 0.25 or 0.5 mgl–1 naphthalenacetic acid (NAA) prior to transfer to green house conditions. This procedure was found to be applicable to seven other cultivars of faba bean from widely diverse provenances. Thus, it can be advantageously applied to the production of transgenic faba bean plants.     

Rapid accumulation of trihydroxy oxylipins and resistance to the bean rust pathogen Uromyces fabae following wounding in Vicia faba

BACKGROUND AND AIMS: Insect damage to plants leads to wound-activated responses directed to healing of damaged tissues, as well as activation of defences to prevent further insect damage. Negative cross-talk exists between the jasmonic acid-based signalling system that is activated upon insect attack and the salicylic acid-based system frequently activated following pathogen infection. Thus, insect attack may compromise the ability of the plant to defend itself against pathogens and vice versa. However, insect herbivory and mechanical wounding have been shown to reduce fungal infections on some plants, although the underlying mechanisms remain to be defined. This work examines the effects of mechanical wounding on rust infection both locally and systemically in the broad bean, Vicia faba and follows changes in oxylipins in wounded leaves and unwounded leaves on wounded plants. METHODS: The lamina of first leaves was wounded by crushing with forceps, and first and second leaves were then inoculated, separately, with the rust Uromyces fabae at various times over a 24 h period. Wounded first leaves and unwounded second leaves were harvested at intervals over a 24 h period and used for analysis of oxylipin profiles. KEY RESULTS Mechanical wounding of first leaves of broad bean led to significantly reduced rust infection in the wounded first leaf as well as the unwounded second leaf. Increased resistance to infection was induced in plants inoculated with rust just 1 h after wounding and was accompanied by rapid and significant accumulation of jasmonic acid and two trihydroxy oxylipins in both wounded first leaves and unwounded second leaves. The two trihydroxy oxylipins were found to possess antifungal properties, reducing germination of rust spores. CONCLUSIONS: These results demonstrate the rapidity with which resistance to pathogen infection can be induced following wounding and provides a possible mechanism by which pathogen infection might be halted.     

Assessment of genetic diversity among faba bean genotypes using agro-morphological and molecular markers

Forty faba bean (Vicia faba L.) genotypes were evaluated for their agro-morphological performance and molecular diversity under Central Region of Saudi Arabia conditions during 2010–11 and 2011–12 seasons. Field performance results showed that faba genotypes exhibited a significant amount of variation for their agro-morphological studied parameters. Giza40 recorded the tallest genotype (139.5 cm), highest number of seeds per plants (100.8), and the highest seed yield per plant (70.8 g). The best performing genotypes were Giza40, FLIP03-014FB, Gazira1 and Goff1. Genetic variability among genotypes was determined using Sequence Related Amplified Polymorphism (SRAP) and Amplified Fragment Length Polymorphism (AFLP) markers. A total of 183 amplified fragments (alleles) and 1758 polymorphic fragments (bands) in SRAP and 202 alleles and 716 bands in AFLP were obtained using six SRAP and four AFLP primer combinations respectively. Polymorphism information content (PIC) values for AFLP and SRAP markers were higher than 0.8, indicating the existence of a considerable amount of genetic diversity among faba tested genotypes. The UPGMA based clustering of faba genotypes was largely based on origin and/or genetic background. Result of cluster analysis based on SRAP showed weak and not significant correlation while, it was highly significant based on AFLP analysis with agro-morphological characters (r = 0.01, p > 0.54 and r = 0.26, p < 0.004 respectively). Combined SRAP and AFLP markers proved to be significantly useful for genetic diversity assessment at molecular level. They exhibited high discrimination power, and were able to distinguish the faba bean genotypes with high efficiency and accuracy levels.     

SSR analysis of genetic diversity and structure of the germplasm of faba bean (Vicia faba L.)

Assessing the diversity and genetic structure of faba bean (Vicia faba L.) germplasm is essential to improve the quality and yield of this economically important crop. In this study, simple sequence repeats (SSRs) were utilized to evaluate the diversity and structure of 35 faba bean genotypes originating from three different geographical regions (Northern Africa, Eastern Africa, and Near East). All 15 SSR loci generated a total of 100 alleles. The allele number per locus varied from 4 to 11, with a mean of 6.67. The expected heterozygosity (H_e) of SSR loci ranged between 0.51 and 0.81, with a mean of 0.63. The PIC value also varied from 0.44 to 0.78, with an average of 0.58. The expected heterozygosity of 22 faba bean genotypes was higher than the observed one. Interestingly, AMOVA analysis showed that much of variability resided within accessions (79.2%). A highly significant difference among regions was also evidenced, and represented 5.3% of the total variation. Moreover, cluster analysis divided the 35 faba bean genotypes into two main clusters. The first main cluster comprised all faba bean genotypes originating from the Near East region, whereas the second main cluster comprised all the genotypes originating from the Northern and Eastern Africa regions, indicating that the Northern and Eastern African faba bean genotypes were more closely related to each other than to the Near East genotypes. Structure analysis also revealed that the 35 faba bean genotypes might be assigned to two populations, in complete accordance with cluster analysis data. In conclusion, this study showed high levels of diversity in the analysed genotypes of faba bean, and could be utilized in future breeding programmes to develop new cultivars of high yield.     

Influence of plant extracts and microbioagents on physiological traits of faba bean infected with<Emphasis Type="Italic">Botrytis fabae</Emphasis>

Laboratory and greenhouse experiments were conducted to assess the efficacy ofEucalyptus citriodora, Ipomoea carnea, Cuminum cyminum, Allium sativum and Hyoscyamus muticus leaf extracts, and Streptomyces exfloliatus (S) andTrichoderma harzianum (T) for controllingBotrytis fabae causing chocolate spot disease of faba bean. Laboratory study supported the use ofE. citriodora (Ex 1) andI. carnea (Ex 2) extracts than the others for controlling the growth ofB. fabae. S+T was the best for inhibiting spore germination followed by Ex 1 +Ex 2 after 8 h of testing, whereas Ex 1 +Ex 2 produced the lowest percent of germination after 16 h. After 4 days, the inhibiting order of the growth ofB. fabae was S+T > Ex 1 +Ex 2 > T > Ex 2 > Ex 1 =S. Greenhouse experiments showed the highest activities of peroxidase, catalase and pectinase in the infected plants. These activities were markedly reduced in healthy plants and widely changed by the biocontrol treatments. Applying biocontrol agents to the infected plants increased minerals (N, P, K and Mg), and both Chl biosynthesis and the photosynthetic activity, which in turn led to accumulation of metabolites. This served the plant to resist the detrimental effects ofB. fabae on the plant growth and yield. In this concern, the efficiency of test biocontrol agents seemed to be in the order:T+S > Ex 1 +Ex 2 >T >S > Ex 2 > Ex.     

Partial purification and characterization of NADH-glutamate synthase from faba bean (Vicia faba) root nodules

The NADH-dependent glutamate synthase (EC 1.4.1.14) from the plant fraction of N₂-fixing faba bean (Vicia faba) nodules has been purified 74-fold to a specific activity of about 3 μmol min⁻¹ mg protein⁻¹ with a final yield of 32%. The NADH-GOGAT activity was associated with a single form of the enzyme that behaved as a monomeric protein with a subunit molecular weight of 195 kDa and a native molecular weight from 222 to 236 kDa estimated by gel filtration or PAGE, respectively. The NADH-GOGAT band on SDS-PAGE was cut out and used to produce antibodies. Western blots of SDS-PAGE of crude nodule proteins revealed a 195 kDa polypeptide in root extracts but not in those of leaves or bacteroids. The antiserum also cross-reacted with a polypeptide of camparable molecular weight (195 kDa) from both amide and ureide transporting species legume nodules, indicating that some antigenic epitopes have been conserved between nodule NADH-GOGAT of different species.     

In Vitro and in Vivo Phosphorylation of Stomatal Phosphoenolpyruvate Carboxylase from Vicia faba L.

The in vitro and in vivo phosphorylation of stomatal phosphoenolpyruvate carboxylase [EC 4.1. 1.31] from Vicia faba L. was demonstrated by feeding the concentrated enzyme after 0–70% ammonium sulfate precipitation of guard cell protoplasts with ³²P and subsequent analysis of autoradiograms and Western immunoblots, after SDS-PAGE, of protein samples. The in vitro and in vivo results provide evidence for a radioactive labeling of the two stomatal PEPCase bands (112 and 110 kDa).     

Effects of turgor potentials of epidermal cells neighbouring guard cells on stomatal opening in detached leaf epidermis and intact leaflets of Vicia faba L. (faba bean)

Leaflets of Vicia faba L. (faba bean) were used to determine whether the mechanical forces resulting from the turgor potentials (Φ_p) of the larger epidermal cells neighbouring guard cells play a significant role in regulating stomatal aperture. When Φ_p, of epidermis and Φ_p of bulk leaflet tissue were compared at midday, Φ_p of epidermis were only 15–25% those of bulk leaflet tissue at all but the most negative leaflet water potentials (Φ). When plants were bagged to increase Φ by reducing vapour pressure differences between leaflets and air, Φ_p of bulk leaflet tissue increased to predawn values, but Φ_p, of epidermis increased to only = 20% of predawn values and stomata opened to their widest apertures. Stomatal apertures were positively correlated with Φ_p of bulk leaflet tissue but they were not correlated with Φ_p of epidermis. Reductions in epidermal Φ_p, began predawn, before stomata were open, and reached minimum values at midday, when stomata were open. We conclude that, in Vicia faba, (1) reduction of Φ_p of epidermal cells begins predawn, reducing the counterforce to stomatal opening that would exist if full epidermal turgor were maintained throughout the day, and (2) changes in Φ_p, of leaf epidermal cells do not play a significant role in regulating stomatal aperture.     

Morphological variability in Rhizoctonia solani isolates from different agro-ecological zones of West Bengal,India

Sheath blight caused by Rhizoctonia solani Kühn is one of the most important diseases of rice, resulting in significant yield loss in rice every year. The rice-based intensified cropping system, edapho-climatological and host variations make the disease problem more complicated. However, the incidence and severity of the disease differ from one location to other, one geographical area to other and even differs from country and region wise. The reasons for this disease severity have been attributed to the variation in host genotype, virulence of the pathogen, prevalence of congenial soil physico-chemical and plants’ surrounding environment and cultural practices. Sixty-seven number of isolates of R. solani from rice, 12 no. of R. solani isolates associated with maize, sugarcane, weeds, cabbage, pointed gourd, watermelon, potato, dolichos bean and aparajita were isolated from different agro-ecological region of West Bengal and three no. of isolates of R. oryzae-sativae obtained from Department of Plant Pathology, BCKV, were used in the present study. Cultural and morphological characteristics revealed considerable diversity among the R. solani isolates. Cultural and morphological analysis of WB isolates of rice has indicated that the diversity among the isolates does not correlated with their origin. On the basis of morphological characteristics, R. solani isolates could be easily separated from R. oryzae-sativae isolates. The no. of sclerotia, hyphal length, wt. of sclerotia and mycelial growth rate are the important morphological markers for differentiation of R. oryzae-sativae from R. solani isolates.     

Tissue- and Cell-Specific Distribution of Connexin 32-and Connexin 26-related Proteins from Vicia faba L.

Different tissues (leaf lamina and midveins, epidermis, primary roots, etiolated shoots, hypocotyl, petioles) and protoplasts from mesophyll, guard cells, and petioles of Vicia faba L., obtained from defined developmental stages were analyzed with polyclonal antibodies raised against mouse liver connexins 26 and 32. Immunostaining after treatment with CX 26 antibodies showed two polypeptides of 21 and 16 kD in all tissues examined except for hypocotyl and epidermis. Additionally, a stronger immunoresponse at 40 kD occurred in extracts from leaf material, mesophyll protoplasts, and roots. Incubation with the CX 32 antibodies resulted in an immunoreactive band at 29 kD in mesophyll protoplast samples, and, a very weak band in extracts from leaf material. A 36 kD polypeptide, present in samples prepared from root, etiolated shoots, petioles, and midveins, did not appear in leaf laminas and mesophyll protoplasts. Extracts from guard cells did not show any immunoreactive band. The tissue- and cell-specific distribution of CX 23- and CX 26-related proteins is supposed to reflect diversities in physiological functions together with alterations in plasmodesmatal ultrastructures during development.     

Growth rate and mitotic index analysis of Vicia faba L. roots exposed to 60-Hz electric fields

Growth, mitotic index, and growth rate recovery were determined for Vicia faba L. roots exposed to 60-Hz electric fields of 200, 290, and 360 V/m in an aqueous inorganic nutrient medium (conductivity 0.07-0.09 S/m). Root growth rate decreased in proportion to the increasing strength; the electric field threshold for a growth rate effect was about 230 V/m. The induced transmembrane potential at the threshold exposure was about 4-7 mV. The mitotic index was not affected by an electric field exposure sufficient to reduce root growth rate to about 35% of control. Root growth rate recovery from 31-96% of control occurred in 4 days after cessation of the 360 V/m exposure. The results support the postulate that the site of action of the applied electric fields is the cell membrane.     

Phylogeny and genetic diversity of native rhizobia nodulating common bean (Phaseolus vulgaris L.) in Ethiopia

The diversity and phylogeny of 32 rhizobial strains isolated from nodules of common bean plants grown on 30 sites in Ethiopia were examined using AFLP fingerprinting and MLSA. Based on cluster analysis of AFLP fingerprints, test strains were grouped into six genomic clusters and six single positions. In a tree built from concatenated sequences of recA, glnII, rpoB and partial 16S rRNA genes, the strains were distributed into seven monophyletic groups. The strains in the groups B, D, E, G1 and G2 could be classified as Rhizobium phaseoli, R. etli, R. giardinii, Agrobacterium tumefaciens complex and A. radiobacter, respectively, whereas the strains in group C appeared to represent a novel species. R. phaseoli, R. etli, and the novel group were the major bean nodulating rhizobia in Ethiopia. The strains in group A were linked to R. leguminosarum species lineages but not resolved. Based on recA, rpoB and 16S rRNA genes sequences analysis, a single test strain was assigned as R. leucaenae. In the nodC tree the strains belonging to the major nodulating groups were clustered into two closely linked clades. They also had almost identical nifH gene sequences. The phylogenies of nodC and nifH genes of the strains belonging to R. leguminosarum, R. phaseoli, R. etli and the putative new species (collectively called R. leguminosarum species complex) were not consistent with the housekeeping genes, suggesting symbiotic genes have a common origin which is different from the core genome of the species and indicative of horizontal gene transfer among these rhizobia.     

Mean, genetic variance, and usefulness of selfing progenies from intra- and inter-pool crosses in faba beans (Vicia faba L.) and their prediction from parental parameters

 Determining the genetic potential of a base population from the properties of their parental lines would improve the efficiency of a breeding program. In the present study, we investigated whether the means of the parents and the genetic distance determined from RAPD data (GD) or multivariate analysis (Mahalanobis D²), mid-parent heterosis (MPH), and the absolute difference between means of the parents (∣P₁−P₂∣) can be used for predicting the means and genetic variances (σ^² _g ) of F_3:4 lines derived from different crosses in faba beans. The material comprised 18 intra- and 18 inter-pool crosses among lines from the Minor, Major, and Mediterranean germplasm pools. Fifty F_3:4 lines from each cross were evaluated for days to anthesis, plant height, seeds per plant, and seed yield in German (GE) and Mediterranean (ME) environments. GD estimates between parent lines ranged from 0.38 to 0.58, while D² ranged from 45.5 to 134.7. Correlations between means of the parents and F_3:4 lines were highly significant for most traits. Estimates of σ² _g for all traits showed non-significant correlations with MPH, GD, D². In one ME, ∣P₁−P₂∣ had significant associations with σ^² _g for seed yield and days to anthesis. The predicted usefulness of crosses, defined as the sum of the population mean and selection responses, was most closely associated with the means of F_3:4 lines. We conclude from this study that the means of F_3:4 lines can be predicted from the means of the parents, whereas the prediction of genetic variance is still an unsolved problem Received: 12 December 1997 / Accepted: 13 July 1998     

The combined effects of CO2 concentration and solar UV-B radiation on faba bean grown in open-top chambers

The response of faba bean seedlings to the combined effects of increased atmospheric CO2 concentrations ([CO2]) and solar UV-B irradiance was studied using open-top chambers transparent to UV-B radiation. The purpose of the study was to determine whether effects of increased [CO2] on growth and physiology are modified by the present solar UV-B fluence rate in the Netherlands. Seedlings were exposed to 350 or 700 micromoles mol-1 CO2. At both [CO2], solar UV-B irradiance was either present or reduced using polyester foil opaque to UV-B radiation. To obtain information on the time dependence of increased [CO2] and UV-B radiation effects, three harvests were performed during the experiment. CO2 enrichment resulted in increased biomass production at all harvests. At the final harvest, UV-B radiation did not affect biomass production but a significant decrease was observed after 14 d of treatment. A reduction of the UV-B fluence increased shoot length at both [CO2] throughout the experiment. UV-B radiation slightly altered biomass allocation. Plants grown at reduced levels of UV-B radiation invested less biomass in flowers and more in stem material compared to plants grown at ambient UV-B levels. CO2 enrichment resulted in a stimulation of net photosynthesis after 26 and 38 d of treatment. UV-B reduction did not alter this response. After 26 d of treatment, photosynthetic acclimation to CO2 enrichment was observed, which was probably the result of accumulation of carbohydrates in the leaves. After 38 d, photosynthetic acclimation was no longer present. The UV absorbance of methanolic leaf extracts was increased by CO2 enrichment only. Both CO2 enrichment and solar UV-B reduced the transmittance of radiation through intact attached leaves. Interaction between [CO2] and UV-B radiation was limited to UV-A transmittance of leaves. Under prevalent experimental conditions, UV-B radiation did not affect the measured physiological parameters. Most open-top chambers used for climate change research are constructed of materials which do not transmit UV-B radiation. Our results indicate that part of the 'chamber effects' on plant height often described in the literature might be explained by the absence of solar UV-B radiation in these chambers.     

Assessment and comparison of AFLP and SSR based molecular genetic diversity in Indian isolates of Ascochyta rabiei, a causal agent of Ascochyta blight in chickpea (Cicer arietinum L.)

Ascochyta blight (AB), caused by Ascochyta rabiei (Pass.) Labr. (anamorph), is the most damaging disease of chickpea (Cicer arietinum L.) and is a serious biotic stress constraint for chickpea production. To understand the molecular diversity in A. rabiei populations of India, a total of 64 isolates collected from AB-infected chickpea plants from different agroclimatic regions in the North Western Plain Zone (NWPZ) of India were analyzed with 11 AFLP (amplified fragment length polymorphism) and 20 SSR (simple sequence repeat) markers. A total of 9 polymorphic AFLP primer pairs provided a total of 317 fragments, of which 130 were polymorphic and showed an average PIC value 0.28. Of the SSR markers, 12 showed polymorphism and provided a total of 29 alleles with an average PIC value 0.35. To the best of our knowledge, this is the first report on a comparison of AFLP and SSR diversity estimates in A. rabiei populations. The dendrogram developed based on AFLP and SSR data separately, as well as on the combined marker dataset, grouped the majority of AB isolates as per geographic regions. Model based population structure analysis revealed four distinct populations with varying levels of ancestral admixtures among 64 isolates studied. Interestingly, several AFLP primer combinations and SSR markers showed the locus/allele specific to AB isolates of certain regions, e.g., Hisar, Sriganganagar, Gurdaspur, and Sundarnagar. Genetic variability present in AB isolates of the NWPZ of India suggests the continuous monitoring of changes in A. rabiei population to anticipate the breakdown of AB resistance in chickpea cultivars grown in India.     

SSR and AFLP based genetic diversity of soybean germplasm differing in photoperiod sensitivity

Forty-four soybean genotypes with different photoperiod response were selected after screening of 1000 soybean accessions under artificial condition and were profiled using 40 SSR and 5 AFLP primer pairs. The average polymorphism information content (PIC) for SSR and AFLP marker systems was 0.507 and 0.120, respectively. Clustering of genotypes was done using UPGMA method for SSR and AFLP and correlation was 0.337 and 0.504, respectively. Mantel's correlation coefficients between Jaccard's similarity coefficient and the cophenetic values were fairly high in both the marker systems (SSR = 0.924; AFLP = 0.958) indicating very good fit for the clustering pattern. UPGMA based cluster analysis classified soybean genotypes into four major groups with fairly moderate bootstrap support. These major clusters corresponded with the photoperiod response and place of origin. The results indicate that the photoperiod insensitive genotypes, 11/2/1939 (EC 325097) and MACS 330 would be better choice for broadening the genetic base of soybean for this trait.     

我国蚕豆根瘤菌的数值分类及其16SrDNA PCR-RFLP研究

对分离自我国11个省24个地区49株蚕豆根瘤菌及11株参比菌株进行了唯一碳源、氮源、抗生素、耐逆性和酶活性等138个表型性状测定,并用MINTS软件进行聚类分析。结果表明,全部供试菌株在59％的相似水平上聚在一起,在80％的相似水平上可分为6个群。其中群4与参比菌株聚在一起,而其他5个群均由未知菌组成。进一步对36株菌进行了16S rDNA PCR—RFLP分析,在85％相似水平上供试菌可分为4个群和1个独立的分支,其聚群结果与数值分类结果有较好的一致性。表型及遗传型分析结果表明,我国蚕豆根瘤菌具有极大的多样性。     

Osmotic regulation of assimilate unloading from seed coats of Vicia faba. Assimilate partitioning to and within attached seed coats.

The significance of the osmotic potential of the seed apoplast sap as a regulator of assimilate transfer to and within coats of developing seed of Vicia faba (cv. Coles Prolific) was assessed using attached empty seed coats and intact developing seed. Following surgical removal of the embryos, through windows cut in the pod walls and underlying seed coats, the resulting attached “empty” seed coats were filled with solutions of known osmotic potentials (–0. 02 versus –0. 75 MPa). Sucrose efflux from the coats was elevated at the higher osmotic potential (high osmotic concentration) for the first 190 min of exchange. Thereafter, this efflux was depressed relative to efflux from coats exposed to the low osmotic potential (high osmotic concentration) solution. This subsequent reversal in efflux was attributable to an enhanced diminution of the coat sucrose pools at the high external osmotic potential. Indeed, when expressed as a proportion of the current sucrose pool size, relative efflux remained elevated for coats exposed to the high osmotic potential solution. Measurement of potassium and sucrose fluxes to and from their respective pools in the coat tissues demonstrated that the principal, fluxes, sensitive to variative in the external osmotic potential, were phloem import into and efflux from the “empty” coats. Phloem import, consistent with a pressure-driven phloem transport mechanism, responded inversely with changes in the external osmotic potential. In contrast, sucrose and potassium efflux from the coats exhibited a positive dependence on the osmotic potential. Growth rates of whole seed were approximately doubled by enclosing selected pods in water jackets held at temperatures of 25°C. compared to 15°C. The osmotic potential of sap collected from the seed apoplast remained constant and independent of the temperature-induced changes in seed growth rates and hence phloem import. Based on these findings, it is proposed that control of phloem import by changes in the external osmotic potential observed with “empty” seed coats has no significance as a regulator of assimilate import by intact seed. Rather, maintenance of the seed apoplast osmotic potential, independent of seed growth rate, suggests that the observed osmotic regulation of efflux from the coats may play a key role in integrating assimilate demand by the embryo with phloem import.