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

2000 ◽  
Vol 6 (4) ◽  
Author(s):  
E. Eissa Ahmed ◽  
Gy. Bisztray ◽  
I. Velich
Keyword(s):  
Phaseolus Vulgaris ◽  
High Velocity ◽  
Callus Tissue ◽  
Marker Gene ◽  
Transient Gene Expression ◽  
Dna Delivery ◽  
Callus Cultures ◽  
Physical Factors ◽  
Foreign Dna ◽  
High Level

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.

scholarly journals Bean tissue culture and genetic transformation with Agrobacterium

2000 ◽  
Vol 6 (1) ◽  
Author(s):  
E. Eissa Ahmed ◽  
Gy. Bisztray ◽  
I. Velich
Keyword(s):  
Transient Gene Expression ◽  
Callus Cultures ◽  
In Vitro Assays ◽  
Gus Gene ◽  
Blue Colour ◽  
Histochemical Assay ◽  
Foreign Dna ◽  
Screenable Marker ◽  
High Level

In this paper we report the establishment methods of a rapidly growing callus culture of Phaseolus vulgaris bean as well as the conditions required for a high level of transient gene expression using Agrobacterium-mediated transformation. A vector is containing both the lindan-resistance gene as a selectable marker, and GUS gene as a screenable marker. By using hypocotyl explant and vertical culture on B5 medium supplemented with 1 mg/1 kinetin- and 2,4-D 2 mg/1 and subcultured every 3-4 weeks, we can recommend to get a good and much callus from bean. This will help in introducing foreign DNA into callus cells. One strain of Agrobacterium carrying plasmid as vector for introducing foreign DNA into plant cells was used. At different concentrations of lindan; 3, 4 and 4.5 mg/I, the transformed Maxidor callus survived and grew over a period of 6 month and subcultured every 3-4 weeks, but the control callus died. Callus were assayed for GUS activity to confirm the expression of the GUS gene using the histochemical assay test. The GUS gene was also correctly expressed in callus cultures grown on 4mg/I lindan-selected medium, the typical blue colour in the histochemical assay using the X-gluc as substrate. But the control, non-transformed callus was not able to grow in the presence of lindan, neither showed a positive reaction in the in vitro assays.

scholarly journals Genetic transformation of bean callus via Agrobacterium- mediated DNA transfer

2003 ◽  
Vol 9 (3-4) ◽  
Author(s):  
E. Eissa Ahmed
Keyword(s):  
Southern Blotting ◽  
Southern Hybridization ◽  
Marker Gene ◽  
Transient Gene Expression ◽  
Callus Cultures ◽  
Hybridization Technique ◽  
Coding Region ◽  
Herbicide Resistant ◽  
Gusa Gene ◽  
Media Data

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.

scholarly journals Biolistic-mediated transient gene expression in shoot apical meristems of the prickly-pear (Opuntia ficus-indica)

1999 ◽  
Vol 42 (3) ◽  
pp. 299-302 ◽  
Author(s):  
Romulo Marino Llamoca-Zárate ◽  
Luiz Ferreira Aguiar Ponte ◽  
Joerg Landsmann ◽  
Francisco de Assis Paiva Campos
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Transient Gene Expression ◽  
Dna Delivery ◽  
Prickly Pear ◽  
Target Tissue ◽  
Gus Gene ◽  
Transformation System ◽  
Opuntia Ficus Indica ◽  
Apical Meristems

We have demonstrated the transient expression of the GUS gene in cells of the meristematic apical dome of Opuntia ficus-indica. DNA delivery into the cells was achieved using a biolistic PDS-1000He instrument from Bio-Rad Laboratories. The transforming DNA was coated in tungsten particles with diameter of 1.3 m m and the distance between the flying disk and the target tissue was 7.5cm and the shooting pressure was adjusted to 1200 psi. This is the first demonstration that the biolistic transformation system can be used to express a transgene in a member of the Cactaceae.

scholarly journals Use of interracial hybridization in breeding the race Durango common bean

1993 ◽  
Vol 73 (3) ◽  
pp. 785-793 ◽  
Author(s):  
Shree P. Singh ◽  
Albeiro Molina ◽  
Carlos A. Urrea ◽  
J. Ariel Gutiérrez
Keyword(s):  
Disease Resistance ◽  
Phaseolus Vulgaris ◽  
Common Bean ◽  
Mosaic Virus ◽  
Seed Yield ◽  
Block Design ◽  
Bean Common Mosaic Virus ◽  
Mass Selection ◽  
Plant Seed ◽  
High Level

Recently, interracial hybridization was used successfully in breeding common bean (Phaseolus vulgaris L.), but its use has not been adequately documented. Approximately 125 lines with medium-sized seed were selected in the first cycle, mostly from race Durango × race Mesoamerica (both from the Middle American domestication center) single- and multiple-cross populations, for disease resistance and race Durango characteristics. Fifteen of these improved lines, three race Durango control cultivars, and one control cultivar each from races Jalisco and Mesoamerica were evaluated for 3 yr (1989–1991) at three locations in Colombia. A randomized complete block design with three replications was used. Lines were developed using visual mass selection for seed yield and/or resistance to diseases in F2 and F3, followed by single plant harvests in F4 or F5 and seed increases in F6 or F7. Lines resistant to bean common mosaic virus and possessing other desirable traits were yield-tested in F7 or F8. All but two lines outyielded Alteño and Flor de Mayo, the highest yielding control cultivars from races Durango and Jalisco, respectively. Two lines also outyielded Carioca, the race Mesoamerica control cultivar. Improved lines tended to possess higher yield per day. All lines were resistant to bean common mosaic virus and most lines also carried a high level of resistance to anthracnose. Plant, seed, and maturity characteristics of most improved lines were similar to those of race Durango control cultivars. These results support the use of interracial hybridization in improving race Durango common bean. Key words: Common bean, Phaseolus vulgaris, race Durango, interracial populations, seed yield, disease resistance

Acridonepoxidgehalte in Kalluskulturen von Ruta graveolens und ihre Steigerung durch Mischkultur mit Pilzen. / Accumulation of Acridone-Epoxides in Callus Cultures of Ruta graveolens Increased by Coculture with Not Host-Specific Fungi

1982 ◽  
Vol 37 (7-8) ◽  
pp. 575-583 ◽  
Keyword(s):  
Tissue Culture ◽  
Callus Tissue ◽  
Callus Cultures ◽  
Culture Conditions ◽  
Significant Rise ◽  
Ruta Graveolens ◽  
Secondary Product ◽  
Fungal Elicitors ◽  
Product Accumulation ◽  
Total Disappearance

Abstract The classic attempt to find optimum culture conditions lead to hormonautotrophic cultures and to dark grown cultures. We made the new attempt to rise the yield of rutacridone-epoxides by co­ culture with not hostspecific fungi. Conditions were chosen that only diffusible elicitors could in­ fluence callus tissue. 14 days of coculture mostly caused reduced acridone-epoxide content, growthrate and up to total disappearance of chlorophyll. Within 3 - 7 days of coculture some fungi induced a significant rise of acridone-epoxide content (up to 50-fold). Culture filtrates of these fungi showed comparable effects. Our results proved the acridone-epoxides to be phytoalexins. The use of fungal elicitors could be a new method to increase secondary product accumulation in tissue culture.

The Influence of Physicochemical Parameters on the Efficacy of Non-Viral DNA Transfection Complexes: A Comparative Study

2006 ◽  
Vol 6 (9) ◽  
pp. 2776-2782 ◽  
Author(s):  
Carsten Kneuer ◽  
Carsten Ehrhardt ◽  
Heike Bakowsky ◽  
M. N. V. Ravi Kumar ◽  
Volker Oberle ◽  
...  
Keyword(s):  
Zeta Potential ◽  
Mammalian Cells ◽  
Transfection Efficiency ◽  
Dna Delivery ◽  
Dna Transfection ◽  
Dna Complexes ◽  
General Validity ◽  
Delivery Vehicles ◽  
Foreign Dna ◽  
Pronounced Reduction

Various polycationic vehicles have been developed to facilitate the transfer of foreign DNA into mammalian cells. Structure-activity studies suggested that biophysical properties, such as size, charge, and morphology of the resulting DNA complexes determine transfection efficiency within one class of vector. To investigate the general validity of these criteria, we studied the efficacy of a variety of DNA delivery vehicles including liposomes (DOTAP, SAINT2) with and without helper lipid (DOPE), the polymer polyethyleneimine (PEI), and cationic nanoparticles (Si26H, PLGA/chitosan) in a comparative manner. Sizes of the DNA complexes varied between 100 and 500 nm for PEI polyplexes and DOTAP/DOPE lipoplexes, respectively. The zeta potential was positive for PEI, Si26H, and DOTAP based complexes, while it was neutral for SAINT2-DNA complexes and negative for PLGA/chitosan-DNA complexes. The latter finding was elucidated by AFM, showing a layer of DNA adsorbed onto the nanoparticles. Transfection activity was negligible for PLGA/chitosan nanospheres, moderate for Si26H nanospheres and high for all other complexes, PEI being the most active carrier. The liposomal preparations were of low (DOTAP) or moderate (SAINT2) stability in serum, resulting in a pronounced reduction of gene expression, which was partially restored by the addition of chloroquine. In conclusion, transfection efficiency (i) seems to require a positive or neutral zeta potential, (ii) is depending on size, e.g., is higher for smaller particles, and (iii) requires a vector that is stable in serum.

Improvement of Vindoline Production in a Catharanthus roseus callus Line

2011 ◽  
Vol 345 ◽  
pp. 375-380
Author(s):  
Bei Bei Xiang ◽  
Ye Rong Zhu ◽  
Wen Juan Wang ◽  
Yan Ling Bai ◽  
Yong Wang
Keyword(s):  
Chlorophyll Content ◽  
Catharanthus Roseus ◽  
Chloroplast Development ◽  
Genetic Manipulation ◽  
Callus Cultures ◽  
Antitumor Drugs ◽  
Callus Line ◽  
Total Alkaloids ◽  
Commercial Value ◽  
High Level

The influence of polyploidity on the accumulation of vindoline in Catharanthus roseus callus cultures was investigated. The callus line (T1) was induced from a tetraploid leaf. Total alkaloids in the callus were extracted and analyzed by LC-MS for qualitification and HPLC for quantification. Results showed that T1 callus cultures could accumulate vindoline to a high level. The highest accumulation of vindoline found in the callus was 0.11 mg g−1 DW. Our results demonstrate that polyploidity could influence the chlorophyll content or chloroplast development and improve vindoline biosynthesis in callus cultures. The T1 callus cultures also accumulated a substance which was not present in the diploid callus. The substance was preliminarily identified by LC-MS as deacetylvindoline, the direct precursor of vindoline biosynthesis. T1 callus could be used for genetic manipulation of the biosynthesis of vinblastine and vincristine, the two important antitumor drugs, and therefore, have potential commercial value.

Isoenzymes in cell cultures of bush bean (Phaseolus vulgaris cv. Contender): isoenzymatic changes during the callus culture cycle and differences between stock cultures

1974 ◽  
Vol 52 (12) ◽  
pp. 2621-2629 ◽  
Author(s):  
Paul G. Arnison ◽  
W. G. Boll
Keyword(s):  
Phaseolus Vulgaris ◽  
Polyphenol Oxidase ◽  
Callus Culture ◽  
Cell Cultures ◽  
Callus Cultures ◽  
Bush Bean ◽  
Leucine Amino Peptidase ◽  
Culture Cycle ◽  
Enzyme Patterns ◽  
Isoenzyme Patterns

Electrophoretic analyses of isoenzyme patterns were performed with extracts of root, hypocotyl, and cotyledon callus cultures derived from a single seedling. The enzymes studied included peroxidase, polyphenol oxidase, catalase, malate and glutamate dehydrogenases, esterase, and leucine amino peptidase. Enzyme patterns changed during the culture cycle and several isoenzymes appeared only at certain times. The isoenzymatic patterns of the three cultures were very similar but persistent differences between them were observed.

scholarly journals Callus Cultures Of Beans Infected With Virus As A Model For Testing Antiviral Compaunds

2019 ◽  
Vol 1 (2) ◽  
Author(s):  
Kovalenko Oleksiy ◽  
Kyrychenko Angelina ◽  
Kovalenko Olena
Keyword(s):  
Callus Tissue ◽  
Bean Common Mosaic Virus ◽  
Callus Cultures ◽  
Model System ◽  
Rt Pcr ◽  
Amplification Product ◽  
Proposed Model ◽  
Plant Tumors

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.

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