GENERATION OF TRANSGENIC MARKER-FREE CUCUMBER PLANTS BY CO-TRANSFORMATION STRATEGY
Abstract
Generating of selectable marker-free transgenic plants is desirable in such countries around the world including Egypt. In this study, thirty selectable marker-free transgenic T1 lines of cucum-ber were generated through embryogenic calli-driven cotyledon explants and Agro-bacterium-mediated transformation. Eighteen T0 transgenic lines were ob-tained, seven of them were positive for the presence of both constructs (the gus-nptII genes and gfp gene). Whereas, a total number of 11 plants were positive for the presence of the gus-nptII genes. The co-transformation efficiency of the gus-nptII genes and gfp gene ranged from 2% to 4% with a total percentage of 14% and an average of 2.8%. While, the total percentage of transformation efficiency for the gus-nptII genes ranged from 4% to 10% with a total of 36% and an average of 7.2%. The percentage of co-transformation frequency of the gus-nptII-gfp genes in comparison to the percentage of gus-nptII and gus-nptII-gfp genes to-gether ranged from 33.3% to 40% with a total ratio of 38.89 and a main of 39.32%. The seven T0 transgenic lines generated a total of 84 transgenic plants in the T1 progeny. Thirty of these transgenic plants were selectable marker- free having only the gfp gene. In addition to 26 transgenic plants have only the both, gus and antibi-otic resistant gene (nptII) and the remain-ing 28 transgenic lines have both the gus-nptII and gfp genes. The total number of progeny generated from T0 plants per line ranged from 5 to 16 plants. On the other hand, regeneration frequency of the total transgenic plants having gus-nptII and those having gus-nptII-gfp ranged from 4% to 10% with an average of 7.2%. The percentage of marker-free transgenic cu-cumber plants in the T1 generation ranged from 20% to 43.75% with a total percent-age of 35.7%. The gfp segregation ratio ranged from 1.5: 1 to 7: 1 and the total χ2 value was 1.587 and ranged from 0.023 to 1.2. Whereas, the nptII segregation ratio ranged from 1.3: 1 to 4: 1 and the total χ2 value was 5.14 and ranged from 0.03 to 3.0. Comparison of the statistic χ2 value with the tabled value at 0.05 % signifi-cance revealed that there is no significant difference from 3:1 segregation ratio. Fluorescence microscope, Gus staining, PCR and χ2 analysis proved the presence and inheritance of the transgenes and that T1 progeny plants segregated in a Mendelian manner. Our study successful-ly resulted in generation of marker-free transgenic cucumber with higher percent-age through co-transformation strategy.References
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