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Transformation of tobacco plants with virEl gene derived from Agrobacterium tumefaciens pTiA6 and its effect on crown gall tumor formation
53-56.Views:137The VirEl protein plays a key role in the transport of VirE2 protein from the bacterium to the plant cell during crown gall tumor induction by Agrobacterium. The virEl gene of A. tutnefaciens pTiA6 was cloned into the plant transformation vector pTd33 yielding pTd93virEl that was introduced into A. tuniefaciens EHA101 and used for tobacco transformation. The presence of the foreign DNA in the putative transgenic plants was confirmed by PCR analysis. Nine of the 41 transformed plants formed only small tumors following infection with the wild-type A. vitis octopine strain AB3. This property was inherited into the T1 generation. The expression of virEl gene in TI plants was demonstrated by Northern blot analysis.
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Bean tissue culture and genetic transformation with Agrobacterium
32-35.Views:117In 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.
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Production of transgenic carnation with antisense ACS (1-aminocyclopropane44-carboxy late synthase) gene
104-107.Views:165Dianthus chinensis and Dianthus caryophyllus varieties were tested for shoot regeneration from leaf and petal explants and transformed with Agrobacterium tuniefaciens strains (EHA 105 and LBA 4404) harbouring an apple derived ACS cDNA in antisense orientation in order to reduce ethylene production and influence the ethylene dependant traits in carnation. After transformation regenerating shoots were selected on MS medium containing 50-75-100-125-150 mg/1 kanamycin and supplemented with 1 mg/1 BA, 0.2 mg/1 NAA. Transgene integration was proved by PCR analysis with npt II spcific primers followed by Southern hybridisation of DNA isolated from green shoots on medium containing 150 mg/1 kanamycin. Several putative transformants were subjected to RT-PCR in order to examine the npt 11 expression at mRNA level. Both the transformant and the non-transformant plants were potted into glasshouse to observe the effect of changed ethylene production on flowering time, petal senescence and vase life.
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Bacterial diseases of grapevine
45-49.Views:360Grapevines are affected by three major bacterial diseases worldwide, such as bacterial blight (Xylophilus ampelinus), Pierce’s disease (Xylella fastidiosa) and crown gall (Agrobacterium vitis). These bacteria grow in the vascular system of their host, thus they invade and colonize the whole plant, independently on symptom development. Latently infected propagating material is a major factor in their spreading. Therefore the use of bacteria-free planting stock has a basic importance in viticulture. Today several innovative diagnostic methods, mostly based on polymerase chain reaction, are available to detect and identify bacterial pathogens of grapevines. For production of bacteria-free plants, the use hot water treatment followed by establishment of in vitro shoot tip cultures is proposed.
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Genetic engineering of apple (Malus domestica Borkh.) for resistance to fungal diseases using g2ps1 gene from Gerbera hybrida (Asteraceae)
15-12.Views:266In the present study, g2ps1 gene from Gerbera hybrida coding for 2-pyrone synthase which contribute for fungal and insect resistance was used. The aim was to work out an efficient approach of genetic transformation for apple cvs. ‘Golden Delicious’, ‘Royal Gala’ and ‘MM111’, ‘M26’ rootstocks for improving their fungal resistance using genetic engineering techniques. Adventitious shoot formation from leaf pieces of apples studied was achieved using middle leaf segments taken from the youngest leaves from in vitro-grown plants.
Optimum conditions for ‚direct’ shoot organogenesis resulted in high regeneration efficiency of 0%, 95%, 92%, 94% in the studied apples respectively. Putative transgenic shoots could be obtained on MS media with B5 Vitamins, 5.0 mg l-1 BAP, or 2.0 mg l-1 TDZ with 0.2 mg l-1 NAA in the presence of the selection agent “PPT” at 3.0-5.0 mgl-1. Shoot multiplication of transgenic shoots was achieved on: MS + B5 vitamins + 1.0 mg l-1 BAP + 0.3 mg l-1 IBA, 0.2 mg l-1 GA3+1.0 g/l MES+ 30 g/l sucrose + 7.0 g/l Agar, with the selection agent PPT at 5.0 mg l-1 and were subcultured every 4 weeks in order to get sufficient material to confirm transformation of the putative shoots obtained. Six, seven, one and six transgenic clones of the apples studied respectively have been obtained and confirmed by selection on the media containing the selection agent “PPT” and by PCR analysis using the suitable primers in all clones obtained for the presence of the selection” bar gene (447 bp) and the gene-of- interest “g2PS1” (1244 bp), with transformation efficiency of 0.4%, 0.6%, 0.1% and 0.3% respectively. These transgenic clones were multiplied further in vitro in the presence of the selection agent ‘PPT’ and rooted in vitro. Rooted transgenic plantlets were successfully acclimatized and are being kept under-containment conditions according to the biosafety by-law in Syria to evaluate their performance for fungal resistance . -
In vitro regeneration from cotyledons of watermelon
96-98.Views:162Cotyledonary segments of five different genotypes of watermelon were used to induce organogenesis. Five different hormone combinations were applied to enhance the induction of shoot formation on the surface of the segments. The phases of organogenesis were followed with light and scanning electron microscope. Shoots were obtained after four weeks, then the shoots were transferred to hormone free medium for root induction.
This method of regeneration can be applied in transformation experiments. GUS histochemical assay was made to check the expected success of using Agrobacterium for the transformation.
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The importance of pathogen-free grapevine propagation material in regards to clonal selection and rootstock breeding in Germany
11-13.Views:203High performing propagation material is essential for a reliable and economical production of quality grapes. Apart from genetic aspects pathogen-freedom is of prime importance in propagation material. In particular virus diseases cause major yield and quality losses and reduced longevity. This is also reflected in the current EU legislation, which focuses on the most common and dangerous viruses: GFLV, ArMV, GLRaV-I and GLRaV-III.Apart from these, locally occurring pathogens, e.g. phytoplasms or agrobacterium, are important as well and should not be present in propagation material. There are several ways to develop pathogen-free clones. Starting with already pathogen-free material is certainly the easiest case, but might not be feasible in local varieties with small acreages and limited vine numbers. In these cases the elimination of pathogens is required, either by heat therapy, tissue culture or somatic embryo genesis.
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The Effects of Some Parameters on Agrobacterium-Mediated Transformation in Muskmelon
46-49.Views:170Some parameters involved in Agrobacterium-mediated transformation in muskmelon Hales best (HBS) were studied. Cotyledon explants excised from 3.5-day-old seedlings were co-cultivated with Agrobacterium tumefaciens harbouring binary vectors which contained GUS and BAR genes. After co-cultivation on a low pH medium, explants were transferred to selective medium, with higher pH, containing Claforan and Finale. The medium was changed every two weeks till shoots were induced. All shoots rooted on MS medium supplemented with 0.3 mg/L IBA. These parameters combined as a whole led to successful transformation. The expression of the introduced gene construct was confirmed by GUS staining of shoot segments.
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Production of transgenic carnation with a heterologous 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase bifunctional enzyme cDNA
75-79.Views:111Transgenic carnations were produced with a modified mammalian bifunctional enzyme cDNA coding 6-phosphofructo-2- kinaseffructose 2,6-bisphosphatase. Relative activity of this enzyme determines the fructose 2,6-bisphosphate (fru 2,6-P2) cytosolic concentration. This metabolite — as a signal molecule — is one of the carbohydrate metabolism regulators. The regenerated Dianthus chinensis and Dianthus caryophyllus shoots were selected on MS basal medium containing 150 mg/1 kanamycin. Transgene integration was proven by PCR analysis with cDNA specific primers followed by Southern hybridization of DNA isolated from selected green shoots, which survived on kanamycin containing medium, so 3 D. chinensis and 20 D. caryophyllus transgenic plants were produced. Transgene expression were examined by RT-PCR. Transformed and control plants were potted in glasshouse to evaluate the effect of modified fru 2,6-P2 on development, growth and carbohydrate metabolism.
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Study of different factors of grapevine regeneration systems and genetic transformation
33-36.Views:207The most limitating factor for successful transformation is the absence of high-yielding regeneration protocols. However, the anther-derived embryogenic culture is an optimal technique for genetic transformation and it has been widely applied in many important cultivars, but the necessity of further development of regeneration systems has been proved. We attempted to produce somatic embryos on a wide range of genotypes from various tissues; leaves, petioles, stem segments. We started the examination of grapevine regeneration via organogenesis, succeeded in inducing shoot from the meristematic tissue of the base of bud by testing induction medium contained different concentrations of two types of hormones. To optimize the conditions of the Agrobacterium-mediated transformation, we studied the effectiveness of different Agrobacterium-treatments, the use of antioxidants and the sufficient quantity of kanamycin for selection of transformed cells.
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Genetic transformation of bean callus via Agrobacterium- mediated DNA transfer
49-53.Views:120Callus 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.