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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:267In 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 . -
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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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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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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Study of genetic transformation efficiency via organogenesis and embryogenesis in eggplant (Solanum melongena L. cv. Embti): effects of co-culture, temperature and kanamycin and hygromycin-based selection procedures
15-23.Views:158The effects of kanamycin and hygromycin-based selection and co-culture temperature ranging from 22 to 28 °C upon eggplant transformation efficiency were evaluated. Both morphogenic pathways, somatic embryogenesis and organogenesis, were adopted using cotiledonary and hypocotyl explants, respectively. Somatic embryos were recovered in the presence of both antibiotics, although lesser escapes were observed in hygromycin-supplemented medium. Indeed, selection provided by this antibiotic was more efficient compared to kanamycin, nevertheless, shoot regeneration was not observed with hygromycin. Significant difference on the frequency of cotiledonary explants displaying callus (FEC) was observed as embryogenesis was concerned, although a higher number of embryos was observed in hygromycin selective media. The frequency of explants presenting callus (FEC), embryos (FEE) and shoots or buds (FERG) did not differ statistically for the tested co-culture temperatures, although higher regenerant number was observed at 24 °C.
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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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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.
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In vitro regeneration from cotyledons of watermelon
96-98.Views:163Cotyledonary 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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Aminoglycoside antibiotics affect the in vitro morphogenic response of chrysanthemum and tobacco
93-104.Views:109Broadly the success of genetic transformation of plants requires non-chimeric selection of transformed tissues and its subsequent regeneration. With rare exceptions, most plant transformation protocols still heavily utilize antibiotics for the selection of transgenic cells containing an antibiotic-degrading selectable marker gene. The morphogenic capacity of in vitro chrysanthemum and tobacco stem and leaf explants change with the addition of aminoglycoside antibiotics (AAs). Of 6 antibiotics tested, phytotoxicity occurred at 10-25 and 50-100 pgml-I in chrysanthemum and tobacco explants, respectively, depending on the size of the explant and the timing of application. The presence of light or darkness also had a significant effect. The use of transverse thin cell layers (tTCLs) in conjunction with high initial AA selection levels supported the greatest regeneration of transgenic material (adventitious shoots or callus) and the lowest number of escapes. Flow cytometric analyses demonstrate that regeneration can be predicted in both species, depending on the ploidy level of the callus. Endoreduplication was not observed in chrysanthemum, even at high AA levels, but occurred (8C or more) in tobacco callus, even at low AA concentrations (5-10 pgml-1). The higher the AA level, the greater the DNA degradation and the lower the 2C and 4C values.
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Production of transgenic bean callus via genetic transformation by DNA-coated tungsten particles
43-47.Views:107Callus 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.
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High-velocity microprojectile mediated DNA delivery into Phaseolus vulgaris callus cells
99-102.Views:113We 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.
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Attempting Regeneration from Cultured Cotyledons and Plant Regeneration from Cotyledonary Nodes in Common Bean (Phaseolus vulgaris L.)
57-60.Views:153Dry seeds from two cultivars of common bean (Phaseolus vulgaris L.) were germinated on sterile cotton and sterile deionized distilled water. Cotyledonary node tissue of seedlings were cultured on Murashige and Skoog(MS)-based media supplemented with different combination of N6-benzyl-aminopurine (BAP) and indole-3-acetic acid (IAA), and benzyladenine (BA) and a-naphthaleneacetic acid (NAA). The results revealed that the regeneration percent and the average number of buds and shoots per explant were influenced by the type of explants and exogeneously added hormones. Multiple shoot induction on dry bean cotyledonary node that contain 4-5 mm from cotyledons and hypocotyl on a medium containing full concentration of MS inorganic salts supplemented with 0.5mg/1 BA and 0.1mg/1 NAA was feasible and the method can be applied in transformation experiments.
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Resistance gene Sw-5 of tomato confers resistance to TCSV in Solanum melongena
41-47.Views:137Eggplants transformed with Sw-5 gene, regenerated by organogenesis and somatic embryogenesis, were resistant to the Tomato chlorotic spot virus, while wild plants did present systemic infection. TO plants were selfed and the segregation analysis of T1 and T2 generation indicated the existence of one or more insertion sites. Southern blot analysis confirmed one or two independent insertions in T2 plants. Different lesions associated with the insertion number were observed in TI and T2 plants. T2 plants with two copies displayed faster hypersensitive reactions and characteristic necrotic lesions that contrasted with slower responses and necrotic ring lesions in plants with one copy. These results suggest that the Sw-5 confers resistance to tospovirus in transgenic eggplants and that the resistant phenotype depends on the number of transgene copies.
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Disease progress of apple scab caused by Venturia inaequalis in environmentally friendly growing systems
56-62.Views:186Progression of apple scab epidemic in six apple cultivars, including two current and susceptible (Gala Must, Elstar), two old (Egri Piros, Darusóvári), and two resistant cultivars (Relinda, Releika), were described and analysed in a two-year-study, in two environmental-friendly growing systems (organic and integrated). Curves of disease progress, linear regression analysis of transformed disease incidence data and Area Under Disease Progress Curves (AUDPC) were used to characterise the epidemic processes of the selected cultivars. Cumulative disease progress curves showed continuous but asymmetrical scab development on the moderate or highly susceptible cultivars Gala Must, Elstar and Egri Piros, and on the tolerant or resistant cultivar Darusóvári and Relinda, in both systems. The cultivar Releika showed no symptoms either on fruit or leaf. In linear regression analysis, the best linearisation was given by logistic transformation. Adequate parameters leaf disease incidence rate, of obtained from a regression equation, were higher in the organic system than in the integrated system. Values of AUDPC showed great differences in leaf disease incidences among cultivars and between growing systems. AUDPC gave more differences for comparison of progresses of disease epidemic than growth rate of disease in different systems of disease control. Moreover, the obtained results were compared with similar studies on different pathosystems, and biological interpretations of the analyses are discussed below.
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Down-regulation of ethylene production in carnation (Dianthus Caryphyllus L.) by an apple derived ACC-cDNA
101-104.Views:127Transgenic carnations were produced with an apple derived antisense ACC-synthase cDNA. Transgenic carnation regenerants were potted in glasshouse. All transformed plants showed normal growth and were true-to-type. Ethylene production — measured at full opening stage — lowered by 30-60 %, no plant with 100 % decrease was identified. The vase-life has been observed for 5 years. 38 % of the transformant carnations showed a higher a relative value in days by more than 2 days to 6 days. Twenty six plants were found exhibiting the most marked alterations in the tested trait. In these plants ethylene production decreased by 37-67 %, they have longer vase-life (by 4 days or more). Since the fragrance variety 'Bíbor' was the plant material for genetic modification of vase-life, this trait has been conserved after transformation in spite of the fact that the position of transgene integration cannot be directed.
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Co-transformation of bean callus using high-velocity microprojectiles- mediated DNA transfer
76-78.Views:122We 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.
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Analyses of the pathogen and weather components of disease progress for modeling apple scab epidemics in integrated and organic production systems
101-106.Views:164The pathogen and weather components of apple scab disease progress were analysed in a three-year study, in two environmental-friendly production systems (organic and integrated) on cvs. `Idared', `Jonica' and 'Mutsu'. Linear regression analyses of transformed disease incidence and severity data and "area under the disease progress curves" (AUDPC) were used for the analysis of the pathogen component. To evaluate the role of the weather component in apple scab epidemic, first, the weekly disease increase was determined at a certain week (n). Weekly disease increase was related to rainfall, relative humidity, Mills' wetness period, temperature and interaction between temperature and relative humidity. Five different periods were used in the analyses: i) week (n-1), ii) week n(n-1), iii) week (n-2), iv) week (n-1)(n-2) and v) week n(n-1)(n-2). In the analyses of the pathogen component, the best transformation function was the logistic one. Regression analyses showed that disease growth rates were higher for disease incidence and for the organic production system than for disease severity and for the integrated production system, respectively. Disease growth rates for leaf incidence were higher than fruit incidence on all the three cultivars. AUDPC values showed great differences in both leaf and fruit incidences among cultivars and between the two production systems. The results the of analyses of the weather component showed that the best relationships between disease increase and weather parameters were found for fruit incidence and leaf incidence in week (n-2) in the organic and integrated production systems, respectively. Results also demonstrated that in week n(n-1) temperature played a more important role in the fungus development than the water parameters (relative humidity, rainfall and leaf wetness). Consequently, infection process is significantly dependent on almost all weather parameters, but during the incubation period the most important weather parameter is the temperature. Results were compared with similar studies and biological interpretations of the analyses are discussed.
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Cultivars, marketing and integrated Production of apples in the Czech Republic
87-92.Views:108Development in apple production in the Czech Republic has been adversely effected during a few recent years by escape of capital investment from the agrifood industry (due to privatisation, restitution and transformation). On the other way round the competition on the domestic market is much higher than it was before because of unlimited import of subtropical and tropical fruit and some import of traditional fruit species as well. Therefore fruit growers are very much concerned in any possible means (which are not economically feasible) to be more competitive. One of the most common approach is the use of new cultivars bred in the country which are resistant or tolerant to diseases and offer better quality both for fresh market and processing. Another way for increasing competition ability for fruit growers is the join and mastering of integrated production which also considerably improve the quality of fruit as far as aspects of health are meant.
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Callus induction on standard type Cymbidium cultivars
108-110.Views:119Tissue cultured Cymbidium PLBs (protocormlike body) were used as starting material to induce embryogenic callus which could serve as objects of genetic transformation. We obtained callus using two methods. The first method was culturing the PLB segments for one month in liquid MS medium in the presence of 0.5 mg/1 benzyladenine and 0.05 mg/1 naphtylacetic acid followed by cultivation on the same composition solid medium with 0.5 g/l activated charcoal for an additional month. Callus formation was observed on 30% of the explants. The second way was to propagate the PLB segments on solid MS medium supplemented with 1 mg/1 thidiazuron. In these cultures we also observed callus formation on 20% of the explants.
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Production of transgenic carnation with antisense ACS (1-aminocyclopropane44-carboxy late synthase) gene
104-107.Views:166Dianthus 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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Shoot induction and plant regeneration from cotyledon segments of the muskmelon variety "hógolyó"
61-64.Views:139Cotyledonary segments of the casaba type muskmelon variety "Hógolyó" were used to induce organogenesis. Fifty different hormone combinations were applied to enhance the induction of shoot formation on the edge of the segments. The phases of organogenesis were followed with light- and scanning electron microscope. Shoot induction was achieved with high frequency. The shoots were transferred to hormone free media for root induction. The rooted plantlets were planted out to soil.
NAA was feasible and the method can be applied in transformation experiments.