Aranda-Rocha, Luwbia

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    Desarrollo de mĆ©todos para la transformaciĆ³n genĆ©tica de frijol comĆŗn a travĆ©s de electroporaciĆ³n
    (2007) Aranda-Rocha, Luwbia; Valencia-Chin, Elide; College of Agricultural Sciences; Rodrƭguez, Abner A.; Porch, Timothy G.; Rodrƭguez, Abner A.; MuƱoz, Miguel A.; Department of Agricultural and Biosystems Engineering
    Common bean (Phaseolus vulgaris L.) is the most important legume crop for small farmers worldwide. Weeds, through competition, cause yield reductions in common bean. Through genetic engineering, herbicide resistant lines have been developed in a number of species, e.g. glufosinate (LibertyĀ®) resistance. Common bean transformation has been pursued using biolistic and Agrobacterium methods with little success. The objectives of this study were to develop a more efficient transformation method for common bean. The study utilized two varieties, Tio Canela 75 and Morales. Four pollen germination media were evaluated and both sucrose and calcium concentrations were varied. To determine the optimum voltage for the introduction of the transgene, three dyes (trypan blue, cotton blue, and aniline blue) and the gfp gene were evaluated. Two methods of electroporation, direct electroporation with an electrode and using cuvettes, and three methods of pollination (ā€˜paper,ā€™ ā€˜agarose,ā€™ and ā€˜dropletā€™) were evaluated. The putative transformants were evaluated genotypically, with PCR and Southern Blot, and phenotypically, with glufosinate at 100, 500, and 800 g GA/ha. The results indicate that one of the liquid pollen germination media was superior and that the 10% sucrose concentration gave the best results. Variation in calcium concentration did not have a significant effect on germination. The trypan blue dye, used to determine the best electroporation conditions, was optimized at 0.04% concentration. The selected transformation conditions were 4.0 kV/cm, 50 Ī¼F and 350 Ī©. In total, 1,108 pollinations were completed, 667 with Morales, and 441 with Tio Canela 75, resulting in 80 and 14 pods, respectively. ā€˜Dropletā€™ was the superior pollination method and the ā€˜cuvetteā€™ method was the best electroporation method. After evaluation with PCR, Southern Blot, and glufosinate, it was determined that transformation was not achieved. Key words: Pollen germination, electroporation, genetic engineering, Phaseolus vulgaris.