optimization of gene transformation in pear (pyrus communis l.) cultivars using gus reporter gene
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2012
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Abstract
Nowadays, genetic engineering methods are able to reduce the time of breeding programs for improvement of fruit trees, as well as offering a focused breeding system. The purpose of the present investigation was to study the factors that affect Agrobacterium tumefacience mediated transformation of two pear cultivars: Bartlett and Harrow Delight. Two explants (leaves and axillary shoot meristems) were inoculated with Agrobacterium strain EHA101 harboring the binary vector pBI121 carrying the nptII and gus genes. Factors that effected the regeneration and gus gene expression were bacterial density OD600 (0.2 and 0.6), acetosyringone concentration (100 µM and 250 µM), usage of pluronic F-68 (0% and 0.02%) and co-cultivation time (24, 72 and 120 hours). The results showed that transformation of Bartlett cultivar was more successful than Harrow Delight cultivar. Also, axillary shoot meristem was a good substitute for leaf explant. Results of the optimization of investigated factors showed that in both explants, low concentration of bacteria increased the rate of gene transformation. On the other hand, applying 250 µM acetosyringone could have positive effect on gene transformation in both explants. In this optimization process, pluronic F-68 was recognized as an effective surfactant. Regarding duration of co-cultivation, the shortest treatment (24 hours) showed the highest amount of gene transformation. Expression, presence and integration of transgenic plants were confirmed by histochemical GUS assay, polymerase chain reaction, dot blot and southern blot hybridization.
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dashti12012tuldoptimization
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| Authors | ;S. Dashti1;A. A. Habashi;H. Abdollahi;M. Chamani;M. Jafarkhani Kermani |
| Journal | genes |
| Year | 2012 |
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