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PubMed2005

A genetic linkage map for azuki bean [Vigna angularis (Willd.) Ohwi & Ohashi].

To make progress in genome analysis of azuki bean (Vigna angularis) a genetic linkage map was constructed from a backcross population of (V. nepalensis x V. angularis) x V.angularis consisting of 187 individuals. A total of 486 markers-205 simple sequence repeats (SSRs), 187 amplified fragment length polymorphisms (AFLPs) and 94 restriction fragment length polymorphisms (RFLPs) -were mapped onto 11 linkage groups corresponding to the haploid chromosome number of azuki bean. This map spans a total length of 832.1 cM with an average marker distance of 1.85 cM and is the most saturated map for a Vigna species to date. In addition, RFLP markers from other legumes facilitated finding several orthologous linkage groups based on previously published RFLP linkage maps. Most SSR primers that have been developed from SSR-enriched libraries detected a single locus. The SSR loci identified are distributed throughout the azuki bean genome. This moderately dense linkage map equipped with many SSR markers will be useful for mapping a range of useful traits such as those related to domestication and stress resistance. The mapping population will be used to develop advanced backcross lines for high resolution QTL mapping of these traits.

Chromosome Mapping↗
PubMed2002

Species differences in ligand specificity of auxin-controlled elongation and auxin transport: comparing Zea and Vigna.

The plant hormone auxin affects cell elongation in both roots and shoots. In roots, the predominant action of auxin is to inhibit cell elongation while in shoots auxin, at normal physiological levels, stimulates elongation. The question of whether the primary receptor for auxin is the same in roots and shoots has not been resolved. In addition to its action on cell elongation in roots and shoots, auxin is transported in a polar fashion in both organs. Although auxin transport is well characterized in both roots and shoots, there is relatively little information on the connection, if any, between auxin transport and its action on elongation. In particular, it is not clear whether the protein mediating polar auxin movement is separate from the protein mediating auxin action on cell elongation or whether these two processes might be mediated by one and the same receptor. We examined the identity of the auxin growth receptor in roots and shoots by comparing the response of roots and shoots of the grass Zea mays L. and the legume Vigna mungo L. to indole-3-acetic acid, 2-naphthoxyacetic acid, 4,6-dichloroindoleacetic acid, and 4,7-dichloroindoleacetic acid. We also studied whether or not a single protein might mediate both auxin transport and auxin action by comparing the polar transport of indole-3-acetic acid and 2-naphthoxyacetic acid through segments from Vigna hypocotyls and maize coleoptiles. For all of the assays performed (root elongation, shoot elongation, and polar transport) the action and transport of the auxin derivatives was much greater in the dicots than in the grass species. The preservation of ligand specificity between roots and shoots and the parallels in ligand specificity between auxin transport and auxin action on growth are consistent with the hypothesis that the auxin receptor is the same in roots and shoots and that this protein may mediate auxin efflux as well as auxin action in both organ types.

Biological Transport↗
PubMed2006

High-level constitutive expression in Pichia pastoris and one-step purification of phospholipase D from cowpea (Vigna unguiculata L. Walp).

Phospholipase D (PLD) is one of the main enzymes involved in signal transduction, vesicle trafficking and membrane metabolism processes. Here we describe the heterologous high-yield expression in the yeast Pichia pastoris, one-step purification and characterization of catalytically active PLDalpha from cowpea (Vigna unguiculata L. Walp). Immunoblotting experiments showed that recombinant PLDalpha is recognized by a polyclonal antibody raised against native soybean PLDalpha. A single calcium-dependent octyl-Sepharose chromatography step was used to obtain a highly purified recombinant PLDalpha, as attested by gel electrophoresis, N-terminal amino acid sequence and mass spectrometry data. From 1L of yeast culture medium, about 8 mg of pure recombinant PLDalpha was obtained and the specific activity measured on phosphatidylcholine was 27 micromol/min/mg. Contrary to what was observed previously with Vigna unguiculata PLDalpha expressed in insect cells, no proteolytic degradation of the N-terminal calcium-dependent C2 lipid binding domain was observed here. This functional recombinant PLDalpha should provide a valuable tool for performing detailed studies on the molecular characterization of enzymes as well as structural studies.

Chromatography, Thin Layer↗
PubMed2000

Random amplified polymorphic DNA (RAPD) analysis in Indian mung bean (Vigna radiata (L.) Wilczek) cultivars.

Greengram [Vigna radiata (L.) Wilczek], also known as mung bean, widely cultivated in a large number of countries, is an important pulse crop of Asia and is considered one of the ancestral species of the genus Vigna. Since yields of greengram have remained low across subtropical and tropical Asia, it is important to estimate genetic diversity in existing cultivars in order to see if the lack of genetic variability might be a constraining factor. In this study, 32 Indian cultivars of greengram were subjected to random amplified polymorphic DNA (RAPD) analysis using 21 decamer primers. A total of 267 amplification products were formed at an average of 12.71 per primer with an overall polymorphism of 64%. The extent of polymorphism was moderate to low. Jaccard similarity coefficient values ranged from 0.65 to 0.92. The cluster analysis resulted in mainly three clusters revealing greater homology between cultivars released from the same source. The results of principal components analysis also substantiated this conclusion. The close genetic similarity between the cultivars could be explained due to the high degree of commonness in their pedigrees. The narrow genetic base of the greengram cultivars revealed in the present analysis emphasises the need to exploit the large germplasm collections having diverse morphoagronomic traits in cultivar improvement programs.

Crops, Agricultural↗
PubMed1985

Isolation and characterization of nodulation genes from Bradyrhizobium sp. (Vigna) strain IRc 78.

An 11.76-kilobase-pair (kb) segment of DNA from Bradyrhizobium sp. (Vigna) strain IRc 78 that hybridizes to nodulation genes of Rhizobium meliloti strain 41 was isolated. Hybridization of the 11.76-kb DNA fragment to DNA from other Bradyrhizobium species revealed a high degree of sequence conservation in this region. Transfer of the 11.76-kb segment to nodulation-defective (Nod(-)) mutants of R. meliloti restored their ability to induce nodules on Medicago sativa (alfalfa). Mutants of strain IRc 78 generated by Tn5 mutagenesis of the 11.76-kb segment fell into three classes according to their symbiotic reaction with Vigna unguiculata (cowpea). Class I mutants of strain IRc 78 were unable to induce root-hair curling or to nodulate; class II induced small, ineffective nodules; and class III showed delayed and decreased nodulation with reduction in amount of nitrogen fixed. Furthermore, in contrast to the wild-type strain, class I mutants could not induce nodules on Glycine max (soybean), Cajanus cajan (pigeon pea), or Arachis hypogaea (peanut). This finding suggests a common function of the 11.76-kb region in the infection of host plants by Bradyrhizobium either through root hairs or by "crack entry."

Journal Article↗
PubMed2006

Radiation-induced phenotypic alterations in relation to isozymes and RAPD markers in Vigna radiata (L.) Wilczek.

PURPOSE: The present investigation is aimed at studies on the effects of gamma rays on in vitro and in vivo damage in Vigna radiata. The parameters studied are germination frequency, seedling injury, isozyme alteration and random amplified polymorphic DNA (RAPD) markers. Results obtained are analyzed in the light of modern applications of radiation damage. MATERIALS AND METHODS: Seeds of Vigna radiata were subjected to gamma irradiation with a dose of 20 - 200 Gy. The percent of seedling damage and frequency of germination were determined. Callus samples were produced in vitro and exposed to gamma rays. The irradiated callus samples were processed to extract total protein, and specifically stained for superoxide dismutase (SOD) and peroxidase isozymes. Total genomic DNA was extracted from irradiated callus samples and subjected to random amplified polymorphic DNA analysis using 23 random decamer primers. RESULTS: Gamma irradiation resulted in retardation in seedling height and decrease in germination frequency in a dose dependent manner. Inhibition assay identified variation in response between different isoforms of SOD on radiation exposure. Changes in peroxidase activity were also observed following irradiation. RAPD analysis showed that new bands appeared in the 20 Gy irradiated sample which in the case of some primers showed similarity with the control. The calli irradiated with 50 Gy and 100 Gy of gamma rays was found to have striking resemblance in banding pattern. Callus irradiated at 200 Gy showed maximum damage. DNA damage as revealed by RAPD analysis was reflected in the appearance of new bands with varying molecular weights. CONCLUSION: New isoforms of SOD appeared after irradiation followed by 24 h recovery. Some isoforms of peroxidase reappeared in calli after 24 h recovery. Results of RAPD analysis indicated that the DNA polymorphism was dose dependent.

DNA Damage↗
PubMed1986

Identification of Rhizobium plasmid sequences involved in recognition of Psophocarpus, Vigna, and other legumes.

Symbiotic DNA sequences involved in nodulation by Rhizobium must include genes responsible for recognizing homologous hosts. We sought these genes by mobilizing the symbiotic plasmid of a broad host-range Rhizobium MPIK3030 (= NGR234) that can nodulate Glycine max, Psophocarpus tetragonolobus, Vigna unguiculata, etc., into two Nod- Rhizobium mutants as well as into Agrobacterium tumefaciens. Subsequently, cosmid clones of pMPIK3030a were mobilized into Nod+ Rhizobium that cannot nodulate the chosen hosts. Nodule development was monitored by examining the ultrastructure of nodules formed by the transconjugants. pMPIK3030a could complement Nod- and Nif- deletions in R. leguminosarum and R. meliloti as well as enable A. tumefaciens to nodulate. Three non-overlapping sets of cosmids were found that conferred upon a slow-growing Rhizobium species, as well as on R. loti and R. meliloti, the ability to nodulate Psophocarpus and Vigna, thus pointing to the existence of three sets of host-specificity genes. Recipients harboring these hsn regions had truly broadened host-range since they could nodulate both their original hosts as well as MPIK3030 hosts.

Base Sequence↗
PubMed1980

In Vitro Incorporation of Selenomethionine into Protein by Vigna radiata Polysomes.

Vigna radiata polysomes efficiently incorporated [(75)Se]selenomethionine, [(14)C]methionine, and [(14)C]leucine in vitro. The optimal conditions for translation were determined to be 4.8 millimolar Mg(2+), 182 millimolar K(+), and pH 7.4. The rates of incorporation of [(75)Se]selenomethionine and [(14)C]methionine were similar when measured separately, but [(75)Se]selenomethionine incorporation was 35% less than [(14)C]methionine incorporation when both amino acids were present in equal molar concentrations. Polyacrylamide gel electrophoresis of the hot trichloroacetic acid precipitable translation products demonstrated synthesis of high molecular weight labeled proteins in the presence of [(75)Se]selenomethionine or [(35)S]methionine. No major differences in molecular weights could be detected in the electrophoretic profiles. Utilization of selenomethionine during translation by Vigna radiata polysomes establishes a route for the assimilation of selenomethionine by plants susceptible to selenium toxicity.

Journal Article↗
PubMed1981

Light Requirement for AgNO(3) Inhibition of Ethrel-Induced Leaf Abscission from Cuttings of Vigna radiata.

To obtain information regarding the antiethylene properties and binding site of Ag(+), studies were initiated to define conditions under which Ag(+) does or does not inhibit ethylene action. AgNO(3), applied as a leaf spray, inhibited 2-chloroethylphosphonic acid (Ethrel)-induced leaf abscission from green cuttings of Vigna radiata in white light but lost considerable activity in the dark. In the absence of Ethrel, AgNO(3) stimulated abscission in the dark. When cuttings were dark-aged for 24 hours prior to treatment with AgNO(3) and aged for an additional 24 hours in the dark after treatment, good inhibition of subsequent Ethrel-induced abscission was restored by returning the cuttings to light. However, when dark aging was preceded by far-red irradiation, considerably less inhibition of Ethrel-induced abscission was restored in the light. AgNO(3) was completely inactive on cuttings aged in the dark and treated with Ethrel in the dark. Light is required for the antiethylene activity of AgNO(3) with regard to leaf abscission of Vigna.

Journal Article↗
PubMed1986

Separation and Characterization of Two Endopeptidases from Cotyledons of Germinating Vigna mungo Seeds.

Two major endopeptidases were present in cotyledons of germinating Vigna mungo seeds, as detected by the zymogram after polyacrylamide gel electrophoresis. They were not detectable in cotyledons of dry seeds, but their intensities on the zymogram increased during germination. During incubation of detached cotyledons, however, the activities showed only a slight increase for 5 days. These two endopeptidases could be separated by Sephacryl S-200 column chromatography. One of them was found to be a serine-endopeptidase as judged by phenylmethylsulfonylfluoride and diisopropyl fluorophosphate inhibition. The other was a sulfhydryl-endopeptidase because of its dependency on 2-mercaptoethanol and inhibition by leupeptin, chymostatin, and antipain. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis indicatd that the two endopeptidases digested the Vigna mungo seed globulin subunits at different rates. The serine enzyme digested the 56 kilodalton subunit at first, but the sulfhydryl enzyme digested the 54 kilodalton peptide more efficiently than the 56 kilodalton peptide. The pattern of digestion of globulin by the combination of the serine- and sulfhydryl-endopeptidases was similar to that using crude enzyme extracts.

Journal Article↗
PubMed1987

Elicitation of Necrosis in Vigna unguiculata Walp. by Homogeneous Aspergillus niger Endo-Polygalacturonase and by alpha-d-Galacturonate Oligomers.

Endo-polygalacturonase (PG) was purified from a commercial preparation of Aspergillus niger pectinase by means of carboxymethylcellulose chromatography, preparative isoelectric focusing, and gel permeation through Sephadex G-50. The enzyme was electrophoretically homogeneous and consisted of a single polypeptide chain with a molecular weight of 33,500. The enzyme exhibited a specific activity significantly higher than those of purified polygalacturonases from phytopathogenic fungi. Galacturonate oligomers with a degree of polymerization higher than four appeared quickly as products of the enzymic hydrolysis of Napolygalacturonate. The oligomers were later degraded to di- and monogalacturonate. The homogeneous enzyme and growing mycelium of Aspergillus niger separately elicited a necrotic response in cowpea (Vigna unguiculata Walp.) pods. Heat-inactivated PG and PG inactivated with specific antibodies did not elicit necrosis, suggesting that the catalytic activity of the enzyme is necessary for its function as an elicitor. The PG-released oligosaccharides from Vigna cell wall and the galacturonides with a degree of polymerization greater than four separately elicited necrosis, whereas di- and monogalacturonate did not.

Journal Article↗
PubMed1989

Isoenzymes of Superoxide Dismutase in Nodules of Phaseolus vulgaris L., Pisum sativum L., and Vigna unguiculata (L.) Walp.

The activity and isozymic composition of superoxide dismutase (SOD; EC 1.15.1.1) were determined in nodules of Phaseolus vulgaris L., Pisum sativum L., and Vigna unguiculata (L.) Walp. formed by Rhizobium phaseoll 3622, R. Ieguminosarum 3855, and Bradyrhizobium sp. BR7301, respectively. A Mn-SOD was present in Rhizobium and two in Bradyrhizobium and bacteroids. Nodule mitochondria from all three legume species had a single Mn-SOD with similar relative mobility, whereas the cytosol contained several CuZn-SODs: two in Phaseolus and Pisum, and four in Vigna. In the cytoplasm of V. unguiculata nodules, a Fe-containing SOD was also present, with an electrophoretic mobility between those of CuZn- and Mn-SODs, and an estimated molecular weight of 57,000. Total SOD activity of the soluble fraction of host cells, expressed on a nodule fresh weight basis, exceeded markedly that of bacteroids. Likewise, specific SOD activities of free-living bacteria were superior or equal to those of their symbiotic forms. Soluble extracts of bacteria and bacteroids did not show peroxidase activity (EC 1.11.1.7), but the nodule cell cytoplasm contained diverse peroxidase isozymes which were readily distinguishable from leghemoglobin components by electrophoresis. Data indicated that peroxidases and leghemoglobins did not significantly interfere with SOD localization on gels. Treatment with chloroform-ethanol scarcely affected the isozymic pattern of SODs and peroxidases, and had limited success in the removal of leghemoglobin.

Journal Article↗
PubMed1966

Fine structure of bacteroids in root nodules of Vigna sinensis, Acacia longifolia, Viminaria juncea, and Lupinus angustifolius.

Dart, P. J. (University of Sydney, Sydney, Australia), and F. V. Mercer. Fine structure of bacteroids in root nodules of Vigna sinensis, Acacia longifolia, Viminaria juncea, and Lupinus angustifolius. J. Bacteriol. 91:1314-1319.-In nodules of Vigna sinensis, Acacia longifolia, and Viminaria juncea, membrane envelopes enclose groups of bacteroids. The bacteroids often contain inclusion granules and electron-dense bodies, expand little during development, and retain their rod form with a compact, central nucleoid area. The membrane envelope may persist around bacteroids after host cytoplasm breakdown. In nodules of Lupinus angustifolius, the membrane envelopes enclose only one or two bacteroids, which expand noticeably during development and change from their initial rod structure.

Acacia↗
PubMed2003

The genetic diversity of the Vigna angularis complex in Asia.

A selected set of accessions of components of the azuki bean (Vigna angularis) complex comprising 123 cultivated accessions and 23 wild or weedy accessions from Bhutan, China (including Taiwan), India, Japan, Korea, and Nepal was analyzed using amplified fragment length polymorphism (AFLP) methodology. Using 12 AFLP primer pairs, 580 unambiguous bands were generated, 313 (53.9%) of which were polymorphic among azuki bean accessions. All 580 bands were used to assess phenotypic (band) and genetic (nucleotide) diversity among the 146 azuki bean accessions. The results indicate five major groups of azuki bean germplasm primarily associated with geographic origin of accessions and their status: wild, weedy, or cultivated. These five groups are (i) Himalayan wild, (ii) Nepal-Bhutan cultivated, (iii) Chinese wild, (iv) Taiwan wild - Bhutan cultivated, and (v) northeast Asian accessions. Within the northeast Asian accessions, three subgroups are present. These consist of (v1) Japanese complex - Korean cultivated, (v2) Japanese cultivated, and (v3) Chinese cultivated accessions. The results suggest domestication of azuki bean occurred at least twice, once in the Himalayan region of southern Asia and once in northeast Asia. The remarkable diversity of azuki bean germplasm in the Himalayan region compared with other regions suggests this is a rich source of germplasm for plant breeding. The results suggest there are important gaps in the germplasm collections of azuki bean and its close relatives from various parts of Asia and that specific collecting missions for Vigna germplasm related to azuki bean in the highlands of subtropical Asia are needed.

Analysis of Variance↗
PubMed1981

Regulation of the F-ATPase from mitochondria of Vigna sinensis (L.) Savi cv. Pitiuba by spermine, spermidine, putrescine, Mg2+, Na+, and K+.

Mitochondria from Vigna sinensis (L.) Savi cv. Pitiuba contain the polyamines spermine, spermidine, and putrescine. The membrane-bound F1-ATPase from mitochondria of Vigna sinensis is activated by these polyamines at physiological concentrations. The effect of polyamines on the membrane-bound of F1-ATPase is dependent on the concentrations of Na+, K+, MgATP, and Mg2+. Excess Na+ or K+ prevents the activation of the membrane-bound F1-ATPase by spermine and spermidine, but not by putrescine. The most pronounced effects were observed at low MgATP concentrations in the absence of Na+ and K+. At [MgATP] = 0.08 mM, spermine activation of the membrane-bound F1-ATPase was 130%. The membrane-bound F1-ATPase is slightly activated by Mg2+ at lower concentrations and strongly inhibited by Mg2+ at higher concentrations. Activation as well as inhibition is dependent on the substrate MgATP concentration. Although there is competition between Mg2+ and MgATP, the binding sites for these two ligands are different (pseudocompetitive inhibition). The inhibition of the membrane-bound F1-ATPase can be reversed by polyamines. There is evidence that the binding sites for Mg2+ and polyamines are identical. The F1-ATPase detached from the membrane is neither activated by polyamines nor inhibited by Mg2+. Therefore, the binding sites for Mg2+ and polyamines seem to be localized on the membrane.

Adenosine Triphosphatases↗
PubMed1979

On the differential divisions and preprophase microtubule bands involved in the development of stomata of Vigna sinensis L.

The manifestation of premitotic cell polarity and the resultant structural asymmetry of the differential divisions participating in the development of stomata of Vigna sinensis vary considerably. However, two morphologically distinct types of differential division were distinguished: (a) 'asymmetrical differential divisions', in which the premitotic polarization of the cell, the eccentric position of the nucleus during division and the differences in size and organization of the daughter cells are obvious; and (b) differential divisions in which the above features are inconspicuous or almost absent. The former occur in the ordinary protodermal cells, the latter in some meristemoids. The organization of a sharply demarcated preprophase microtubule band (PMB) precedes, all differential and non-differential divisions. In the first type of differential division the PMB is formed eccentrically, while in the second it may display either an approximately symmetrical or a clearly asymmetrical disposition, always indicating with surprising accuracy the sites where the succeeding cell plate will join the parent walls. The PMB foreshadowing the highly curved cell plates in meristemoids I of the mesoperigenous process, as well as in meristemoids I and II of the mesogenous one, are apposed only on one anticlinal wall and therefore do not encircle the nucleus or traverse the cell. In the symmetrical divisions of guard cell mother cells (GMC), as well as in those of protodermal cells, the PMB runs right round the internal plasmalemma surface in an equatorial position, coinciding with that of the future cell plate. In the former cells the wall abutting the cortical cytoplasm traversed by the band becomes locally thickened. The variability in the pattern of the microtubules of the band along the walls of the GMC is directly mirrored in the pattern of the thickening. It seems that in GMC the PMB mediates a directed exocytosis of dictyosome vesicles. In contrast to what is now generally accepted in dicotyledonous plants, each meristemoid I of both the mesogenous and mesoperigenous stomata in Vigna sinensis leaves does not inhibit but induces the formation of other meristemoids close to it.

Cell Differentiation↗
PubMed2003

Fermentation of Vigna sinensis var. carilla flours by natural microflora and Lactobacillus species.

Natural fermentation and an inoculum containing 10% (vol/vol) Lactobacillus fermentum or Lactobacillus plantarum were used to obtain fermented flours from Vigna sinensis L. var. carilla seeds that had been washed with distilled water and dried at 55 degrees C for 24 h. To optimize the fermentation parameters (lactic acid bacterium level, bean flour concentration, and fermentation time), several small-scale fermentation processes were carried out. On the basis of the results obtained, fermentor-scale bean fermentation by microorganisms present on the seeds (natural fermentation [NF]) or by inoculation with L. plantarum (PF) was carried out at 37 degrees C for 48 h with a concentration of 300 g of bean flour per liter. The fermented flours (NF and PF) were also autoclaved. The levels of alpha-galactosides, inositol phosphates, trypsin inhibitor activity (TIA), soluble carbohydrates, starch (total and available), total available carbohydrates, thiamin, and riboflavin were determined for the processed cowpea flours, and microbiological studies were also carried out. The beans' levels of alpha-galactosides, TIA, and inositol hexaphosphate decreased by 95, 50, and 85%, respectively, for the NF flour and by 87, 27, and 85%, respectively, for the PF flour, while inositol pentaphosphate and inositol tetraphosphate were present in both fermented flours. The sucrose content decreased, and glucose, fructose, and galactose appeared as a result of fermentation. The levels of total available sugars and thiamin decreased by 2 and 12% and by 69 and 43%, respectively, while the riboflavin content increased by 106 and 94% for NF and PF flours, respectively. When NF and PF cowpea flours were heated in an autoclave for 20 min, TIA decreased further (by 80 and 56%, respectively). According to the chemical and microbiological results obtained in this study, fermentation with L. plantarum and autoclaving is an excellent process by which to produce a new functional food from the seed of a cheap legume (Vigna sinensis L. var. carilla).

Carbohydrates↗
PubMed2004

[Use of Phaseolus vulgaris and Vigna sinensis in a fermented dairy drink].

The objective of this work was to develop a new kind fermented dairy drink, partially substituted with clear varieties of Phaseolus vulgaris (caraota) and Vigna sinensis (frijol). The formulation of fermented dairy drinks included sterile extracts of caraota and frijol, as partial substitutes which replaced milk: 10, 20 and 30%. The mixtures were inoculated with 2% of a mixture of Lactobacillus acidophillus, Streptococcus thermophilus and Bifidobacterium sp. and were incubated at 42 degrees C for 7 hours. Mango and guava jams were used as flavorings at 20%. On the basis of the sensorial evaluation the mixtures 10% frijol-mango, 10% frijol-guava, 30% caraota-mango and 20% caraota-guava were selected. In the selected fermented dairy drinks, the levels of protein, soluble and insoluble fiber, available and resistant starches were increased and the protein digestibility was 81%. The technical feasibility of partial substitution of milk with extracts of Phaseolus vulgaris or Vigna sinensis. For the elaboration of a fermented dairy drink similar to the liquid yogurt kind was demonstrated.

Cultured Milk Products↗