Bean Callus Research Articles

Вірусінфікований калюс квасолі та його оздоровлення in vitro за дії ліпосомальних препаратів

The aim of the study was to develop a recovery means for beans infected by Bean yellow mosaic virus (BYMV) as well as Bean common mosaic virus (BCMV) using callus culture and liposomal glycan preparations. Methods. Cultivation of explants and callus cultures was carried out in vitro using conventional methods of plant biotechnology. The tissue culture propagation was performed during the spring or summer seasons. The presence of viral infection was tested by reverse transcription polymerase chain reaction. The virus-specific primers that allowed amplifying the conserved regions of the capsid protein gene of BCMV or BYMV were used for virus identification. Results. The culture of bean callus infected with BCMV was obtained and adapted for antiviral agents testing. It has been shown that during long-term cultivation (10–12 weeks) in the presence of liposomal preparation containing Ganoderma adspersum glucan (10–100 mg/l), plant tissue culture become free from viruses following virus eradication. This is evidenced by the absence in the callus tissue of 391 bp sequences typical for the virus coat protein gene. Conclusions. The full suppression of virus reproduction and gradual elimination of virus occurred in callus tissue obtained from BCMV-infected beans and cultured on B-5 medium supplemented with liposomal glycanglycolipid complex (10–100 mg/l). The data obtained can be useful for the development of practical control method to cure plant virus diseases using callus culture and antiviral-active glycan-glycolipid complexes.

Callus Cultures of Beans Infected With Virus as a Model for Testing Antiviral Compaunds

In the work, bean callus raised from a leaves of Bean common mosaic virus infected bean plant was obtained and adapted for the testing of antiviral activity of liposomal glycan-glycolipid complexes. Ganoderma adspersum glucans and Pseudomonas spec. rhamnolipids were constituents of liposomal compaunds. It has been shown that under the long-term cultivation (up to 3 months) in the presence of a liposomal preparation containing (10-100 mg/l), the virus is eliminated from the tissue. This is evidenced by the absence of 391 bp sequence amplification product established by RT-PCR in the callus tissue, cultured on a medium containing the liposomal complex. The proposed model system is analogous to plant tumors and has obvious advantages over similar systems in vivo, since the callus growth is controlled and independent of environmental factors.

Accumulation and distribution of copper in castor bean (Ricinus communis L.) callus cultures: in vitro

Copper (Cu) accumulation, subcellular distribution and the chemical forms of Cu, and amino acids metabolism were investigated in castor bean (Ricinus communis L.) callus via in vitro culture. The castor bean callus was obtained from the embryo cultured in Murashige and Skoog (MS) medium and then transferred to MS medium with different Cu doses (0, 20, 40, and 60 mg L−1; Cu0, Cu20, etc.) for 28-days cultivation. The stress from Cu inhibited the growth of the castor bean callus, and the Cu content in the castor bean callus increased with the increasing Cu dose, reaching a maximum value of 293.2 mg kg−1 fresh weight (FW) in the 60 mg L−1 Cu treatment. Concentrations of Cu in the cell wall, organelles, and cytoplasm increased significantly with the elevated Cu dose, with the cell wall containing 50.2 % of the total Cu in the 60 mg L−1 Cu treatment. The major Cu fractions were C (bound to the exchangeable polar compounds) (28.1 %) and E (bound to the structural polar compounds and nucleic acids) (27.5 %) in the control treatment, and the main fraction was C (51.2 %) in the treatment with 60 mg L−1 Cu. The concentration of free amino acids in the cytoplasm was closely related to the Cu content in the castor bean callus. In addition, most of the Cu in the cell wall bound with functional groups of the cell chemical components, hydroxyl (–OH), acylamino (–CONH2), and carboxylate ion (–COO−). The castor bean exhibited a strong tolerance to Cu, which accumulated mainly in the cell wall.

Functional characterization of a defense-related class-III chitinase promoter from Lupinus albus, active in legume and monocot tissues

A class-III chitinase promoter was isolated from Lupinus albus. The region 5′ to the coding sequence of the IF3 gene was amplified by gene walking and sequenced. The proximal 2.0 kb sequence contains a predicted promoter site, including a TATA box, near the ATG start site. To test for minimal sequences needed for promoter activity, the region was restricted into fragments of 1.81, 1.51 and 1.13 kb and cloned into the pDM327 vector, upstream from the bar-gus fusion gene for Biolistic™ transformation. Transformation of lupin embryos, bean callus tissue, maize embryos and Ornithogalum callus demonstrated promoter activity for all fragments. In silico analysis identified putative cis-acting elements in the 1.81 kb fragment that could be important in controlling gene expression. Fungal elicitor activated-, wound-inducible- and ethylene responsive elements were present in the 1.51 kb fragment. Myb elements and CAAT boxes that regulate responses to environmental factors and modulate promoter efficiency were identified in the 1.81 kb fragment. The 1.51 and 1.81 kb fragments were inserted upstream of the gus gene into the pBI121 vector for Agrobacterium tumefaciens transformation of tobacco. Quantitative GUS assays indicated that the promoter fragments are functional in planta and inducible by defense-related signals, wounding, as well as chemical elicitation. All important elements essential for Bion inducibility are present on the shorter (1.51 kb) promoter fragment, but both 5′ distal and proximal cis-elements are required for full functionality. The IF3 promoter is, thus, suitable for use in defense gene constructs prepared for the production of anthracnose resistant lupin.

Impact of lectins isolated from the bean callus culture on growing drought tolerance in plants

Plant cell and tissue culture is a very prospective model to detect newsources of lectins, to study different ways of their regulation and to invent new biotechnological methods for isolation them from plants. It canhelp to use lectins as plant protectors in the future. It has been studiedthat lectins obtained from common beans calluses empower drought tolerance of legumes and grains and also positively influence on growingprocesses, water metabolism and prolin content. It has been shown thatlectins stimulate seeds to germinate, impact on plant height and biomass,water in tissues and rate of transpiration. This research can help to makenew biopreparations on the basis of common beans’ lectins to increaseproductivity of crops.Key words: common beans, lectins, water metabolism, drought tolerance, biopreparations

The Phaseolus vulgaris PvTRX1h gene regulates plant hormone biosynthesis in embryogenic callus from common bean.

Common bean is the most important grain legume in the human diet. Bean improvement efforts have been focused on classical breeding techniques because bean is recalcitrant to both somatic embryogenesis and in vitro regeneration. This study was undertaken to better understand the process of somatic embryogenesis in the common bean. We focused on the mechanisms by which somatic embryogenesis in plants is regulated and the interaction of these mechanisms with plant hormones. Specifically, we examined the role of the gene PvTRX1h, an ortholog of a major known histone lysine methyltransferase in plants, in somatic embryo generation. Given the problems with regeneration and transformation, we chose to develop and use regeneration-competent callus that could be successively transformed. Embryogenic calli of common bean were generated and transformed with the PvTRX1hRiA construction to down-regulate, by RNA interference, expression of the PvTRX1h gene. Plant hormone content was measured by mass spectrometry and gene expression was assessed by q-PCR. Detailed histological analysis was performed on selected transgenic embryogenic calli. It was determined that down-regulation of PvTRX1h gene was accompanied by altered concentrations of plant hormones in the calli. PvTRX1h regulated the expression of genes involved in auxin biosynthesis and embryogenic calli in which PvTRX1h was down-regulated were capable of differentiation into somatic embryos. Also, down-regulation of PvTRX1h showed increased transcript abundance of a gene coding for a second histone lysine methyltransferase, PvASHH2h. Accordingly, the PvTRX1h gene is involved in the synthesis of plant hormones in common bean callus. These results shed light on the crosstalk among histone methyltransferases and plant hormone signaling and on gene regulation during somatic embryo generation.

EFFECT OF ALKOHOLIC EXTRACTS OF CHAMOMILE (Matricaria chamomilla L.) CALLUS IN INITIATION, GROWTH AND PROTEIN CONTENT OF BLACK BEAN (Nigella sativa L.)STEMS CALLUS

The study was carried out to know the effect of alkoholic extracts of chamomile (Matricaria chamomilla L.) in callus initiation and growth of explants of black bean (Nigella sativa L.) ,apart from the protein content. Results cleared different response of explant to callus initiation and growth with different concentrations of alkoholic extracts of chamomile . Which had been added to the standard medium of black bean callus initiation and growth ] MS+0.221mg/L 2,4-D (2,4- Dichchlorophenoxy acetic acid) [ .The results refer when adding of 5 mg/ L of chamomile alkoholic extracts to standard MS medium was the best in initiation and growth the callus after 60 days with fresh weight average 12.11 g as comparing with 6.35 g of the standard medium with increasing in total content at 1.21 mg /gm as comparing with 0.61 mg /gm of standard medium ,and when adding chamomile alkoholic extracts alone to MS medium enhanced with degree initiation and growth callus..And also showed that adding of 5 mg /L alkoholic extracts to MS medium greatly enhanced callus initiation and growtg after 60 days with fresh weight average 8.54g with protein content 0.88 mg /gm as comparing with MS medium alone.

The possibility of using Glycyrrhiza glabra L. Callus extracts as an alternative to plant growth regulators in plant tissue culture experiments

Liquorice plant is considered one of the important medicinal and economical plants. It is rich with many compounds, minerals, vitamins, and even plant hormones. This research is aimed to study the possibility of using callus tissue extracts as an alternative to plant growth regulators added to the culture media. A factorial experiment was implemented to find out the appropriate combination between 2, 4-D and BA for callus induction on Liquoricenode explants. It was found that a combination of 2 mg/l 2, 4-D with 2.5 mg/l BA is the best one for callus induction and maintenance using MS medium. Water and alcoholic extracts were prepared from callus tissue at concentrations (0, 2, 4, 6, 8 or 10) ml/l then added to culture medium as an alternative to plant growth regulators. The effect of these concentrations on growth and development of tissues and organs for some plants was studied using soya bean, potato and wheat plants for this purpose. Results showed that water extract induced shoot proliferation from potato single nodes. Both types of extracts increased soya bean callus fresh weight significantly. It was found also that water extract was more effective than alcoholic one in increasing vegetative and root parts in germinating wheat seeds.

Increase in XET activity in bean (Phaseolus vulgaris L.) cells habituated to dichlobenil

Bean (Phaseolus vulgaris L.) cells have been habituated to grow in lethal concentrations of dichlobenil (DCB), a specific inhibitor of cellulose biosynthesis. Bean callus cells were successively cultured in increasing DCB concentrations up to 2 microM. The 2-microM DCB habituated cells were impoverished in cellulose and xyloglucan, had an increased xyloglucan endotransglucosylase (XET; EC 2.4.1.207) activity, together with an increased growth rate and a decreased molecular size of xyloglucan. However, the application of lethal concentrations of two different cellulose-biosynthesis inhibitors (DCB and isoxaben) for a short period of time produced little effect on XET activity and xyloglucan molecular size. We propose that the weakening of plant cell wall provoked by decrease in cellulose content might promote the xyloglucan tethers and increase the ability of xyloglucan to bind to cellulose in order to give rigidity to the wall.

FTIR spectroscopy monitoring of cell wall modifications during the habituation of bean ( Phaseolus vulgaris L.) callus cultures to dichlobenil

The habituation of bean calluses to dichlobenil results from the acquisition of a modified cell wall, with an enhancement in pectins and a decrease in cellulose and hemicelluloses. In this work, Fourier transform infrared (FTIR) spectroscopy in conjunction with a set of multivariate analyses and other statistical tools, such as principal component analyses and Student's t-test applied to clusters from a dendrogram, were used to monitor the modifications occurring in the cell walls of bean callus cultures due to a habituation program to dichlobenil. Forty samples of calluses, differing in the dichlobenil concentration at which they were growing and in the number of subcultures in a given concentration of dichlobenil, corresponding to two habituation experiments, were analyzed. Multivariate analyses of the spectra showed that the type and the extent of cell wall modifications depended on the concentration of the inhibitor in the culture medium and the time that the callus had been present at a given concentration of the inhibitor, and the analyses distinguished among three groups of calluses with different levels of habituation to dichlobenil: (i) non-habituated and habituated to low dichlobenil concentrations, (ii) habituated to intermediate concentrations (up to 4 μM), and (iii) habituated to high concentrations (4–12 μM). A slight modification of cell walls was only detected after 13 subcultures in 0.5 μM dichlobenil. In the presence of a higher concentration of dichlobenil, the content of cellulose was clearly reduced while that of pectins was increased. We conclude that FTIR spectroscopy associated with a set of statistical tools is a powerful method for analyzing in muro–and more rapidly–the changes in polysaccharides related to dichlobenil habituation, and that it could be used in the future to identify cell wall changes related to habituation to other herbicides or stress factors.

Production of transgenic bean callus via genetic transformation by DNA-coated tungsten particles

Callus cultures were induced from hypocotyl of young bean seedlings. The B5 medium completed with 1 mg/1 KIN and 2mg/1 2,4-D proved the best. Callus developed and maintenaned on B5 medium supplemented with 1mg/1 kinetin and 2mg/I 2,4-D. The B5 medium supplemented with 1mg/1 KIN and 2mg/1 2,4-D induced much more callus than half strength MS medium supplemented with 0.5 or 0.75mg/1 BA and 0.1 mg/1 NAA. The results demonstrate that GeneboosterTM is convenient method to obtain transient gene expression in callus of bean. The results have shown that the bean callus shot by GeneboosterTM can be transformed to get (kanamycin-resistant and stress mannitol­tolerant) calli. The presence of mannitol-dehydrogenase gene (mt/) was verified by PCR, showing the integration of mt/ gene carried by two plasmids. Co-transformed calli were selected after bombardment on kanamycin, mannitol and (kanamycin+mannitop-containing media. Data of molecular analysis (PCR) confirmed the insertion of mtl gene in the genome of mannitol-tolerant callus lines.

Genetic transformation of bean callus via Agrobacterium- mediated DNA transfer

Callus cultures were induced from hypocotyl of young bean seedlings. Callus developed and maintenaned on B5 medium supplemented with 2mg/1 2,4-D and 1 mg/1 kinetin. The results demonstrate that A. tumefacins-mediated transformation is a convenient method to obtain transient gene expression in callus of bean. The results have shown that the bean callus co-cultivated with A. tumefaciens can be transformed to get heibicide Finale (glufosinate-ammonium) resistant GUS positive tissues. Southern blot analysis of transformed calli showed integration of gusA marker gene carried by a binary vector. Transformed calli were selected on herbicide containing media. Data of molecular analysis (Southern blotting) confirmed the insertion of gusA gene in the genome of herbicide resistant calli with bar gene. There are three evidences that calli are stable transformants: (1) herbicide resistance, (2) GUS activity which is indicative since the coding region containing an intron, (3) the results of Southern hybridization technique.

Co-transformation of bean callus using high-velocity microprojectiles- mediated DNA transfer

We have found that 50 mg/I kanamycin and 0.8 Mo1/1 mannitol concentration was sufficient to kill the control callus of bean (Phaseolus vulgaris L.) and differentiate transgenic from the non-transgenic cells. The GeneBooster particle delivery system was used for the bombardment of bean callus. The kanamycin resistance gene was used as a selectable marker. The test was made by transferring the healthy white callus, subcultured for three months on selective and non-selective medium. After selection on kanamycin containing media, several kanamycin resistant calli had been obtained, survived and grew. After selection on mannitol containing media no drought resistant calli had been obtained. Resistance of the selected calli were verified by their ability to grow repeatedly on selective medium containing 150 mg/I kanamycin. Selective pressure was maintained over a period of 8 months.

Characterization of cell walls in bean ( Phaseolus vulgaris L.) callus cultures tolerant to dichlobenil

The increase in dry weight during the culture of bean callus cultures was inhibited by the herbicide dichlobenil (2,6-dicholorobenzonitrile) with an I 50 of 0.5 μM. However bean calli became tolerant to a concentration of 12 μM by a stepwise increase in the concentration of the inhibitor in each subculture. Tolerant calli growing in 2,6-dicholorobenzonitrile developed with hollow protuberances. Groups of cells in these protuberances had irregular cell walls surrounded by a thicker cell wall with a lamellate structure and without a differentiated middle lamella. FTIR spectra of tolerant cell walls revealed an increase in both esterified and non-esterified pectins. Cell wall fractionation showed that in tolerant cell walls the xyloglucan–cellulose network of non-tolerant cell walls was partly replaced by a pectin-rich network mainly formed of cross-linked polyuronides with a large proportion of homogalacturonan. These modifications are comparable to those described for bean calli tolerant to isoxaben, pointing to a related mechanism of tolerance for both herbicides.

High-velocity microprojectile mediated DNA delivery into Phaseolus vulgaris callus cells

We report the method for the establishment of rapidly growing callus cultures of Phaseolus vulgaris and the conditions required for efficient transformation using high velocity microprojectiles and high level of transient gene expression. Using hypocotyl explant and vertical culture on B5 medium with lmg/1 kinetin and 2 mg/1 2,4-D, we can recommend to get a rapidly growing callus from bean which is a good starting material to introduce foreign DNA into bean cells. The GeneBooster particle delivery system was used for the bombardment of bean callus and the Hgm resistance gene (Hgmr) was used as a selectable marker gene. 25mg/I hygromycin (Hgm) concentration was sufficient to kill the control callus. We used the standard physical factors, the appropriate pressure of N2 gas for the bombardment of the callus tissue, the shooting distance and the size of tungsten particles used as microprojectiles. Selective and nonselective tests were made by transferring the healthy green and white calluses, subcultured for 4 months on selective and nonselective medium. Several Hgm resistant calli had been obtained. Selective pressure was maintained over a period of 10 months.

Reaction of common bean callus to culture filtrate of Colletotrichum lindemuthianum: Differences in the composition and toxic activity of fungal culture filtrates

Culture filtrate of Colletotrichum lindemuthianum caused dark brown lesions on the lower surface veins of bean leaves. This phenomenon was used as a bioassay to study the production of toxic fungal metabolites. Calli from anthracnose-susceptible bean cultivars 'Collacia', 'Andecha' and 'Seronda' were sensitive to a 12.5% solution of race 38 filtrate or to a 25% solution of race 7 filtrate. In contrast, calli from anthracnose-resistant bean genotypes A 247, TU, PI 207262, 'Collacia' × 'Tu', 'Collacia' × AB 136 and 'Collacia' × PI 207262 did not develop browning. Culture filtrates were passed through an ionic-exchange resin and a gel filtration resin. Toxic activity of fractions from two races of the fungal pathogen was different, although in both races slight necrosis was produced by the same nine fractions. Pathogenicity could be related with common substances and toxicity could be identified with differential compounds.

Cell wall modifications in bean (Phaseolus vulgaris) callus cultures tolerant to isoxaben

Cultures of bean (Phaseolus vulgaris L.) callus were adapted for growth in a 12 μM concentration of the herbicide isoxaben; 1 200‐fold greater than the I50 calculated. Tolerant calluses grew slower, were browner and harder than nontolerant ones and formed large globular corpuscles on their surface. Cell wall fractionation of tolerant cells indicated that the mechanism of adaptation depends on the ability of these cells to divide and expand with a modified wall. The walls of tolerant cells growing on isoxaben differ from those of nontolerant cells by having reduced levels of cellulose and hemicelluloses and a higher uronic acid content. These differences are already found in adaptations to low concentrations of isoxaben and keep to the highest adaptation levels tested without any great change.

Production of Transgenic Kidney Bean Shoots by Electroporation of Intact Cells

We obtained transformed bean shoots by electroporation of intact bean cells with the plasmid pDPG165 containing bar gene conferring herbicide resistance to plants. Transformed shoots were selected from electroporated callus on herbicide containing media. Data of molecular analysis (PCR and Southern blotting) confirmed the insertion of bar gene in the genome of herbicide resistant shoots. Detailed procedures for obtaining regenerative bean callus, optimization of electroporation of intact cells and transgenic shoots are given.

Expression of α-amylase in cultured callus of french bean

α-Amylase (EC 3.2.1.1) expression was found in calli of French bean (Phaseolus vulgaris L. cv Goldstar). We examined enzyme activity in the calli to investigate influence of gibberellin and sugars on enzyme expression. After subculture of the calli, α-amylase activity decreased, and then increased at a stationary phase of callus growth. Exogenous application of gibberellin and an inhibitor of gibberellin synthesis, uniconazole, did not have any significant effects on the enzyme expression. Sugar starvation increased the activity, while addition of metabolizable sugars, such as sucrose, glucose and maltose, to the medium repressed expression. Addition of 6% mannitol, a non-metabolizable sugar, to the medium induced higher α-amylase expression as compared to addition of 3% mannitol. This result suggests that osmotic stress enhances α-amylase activity in the calli. Furthermore, high concentrations of agar in the medium increased α-amylase activity in the calli. It is probable that high concentrations of agar prevented incorporation of nutrient into the calli and induced the α-amylase activity in the calli.

Somatic embryogenesis and plant regeneration from leaf derived callus of winged bean [ Psophocarpus tetragonolobus (L.) DC.

Somatic embryogenesis and plant regeneration from leaf derived callus of winged bean [ Psophocarpus tetragonolobus (L.) DC.