Agrobacterium-Mediated Insect Resistance Gene (cry1Aa) Transfer Studies Pertaining to Antibiotic Sensitivity on Cultured Tissues of Broccoli

Abstract

ABSTRACTSuccessful plant transformation requires an efficient regeneration protocol and a suitable selection method. A genetically engineered, disarmed Agrobacterium tumefaciens strain containing a binary vector pBin-1Aa with cry1Aa (insect resistance) and npt-II (neomycin phosphotransferase-II) genes was used for genetic transformation studies. Effects of antibiotics kanamycin and cefotaxime were studied to determine the aptness of kanamycin resistance as a selectable marker and for cefotaxime in controlling excessive bacterial growth during genetic transformation studies using cultured hypocotyl, cotyledon, leaf, and petiole tissues of Broccoli (Brassica oleracea L var. italica). Leaf and petiole explants exhibited decreased fresh weight as kanamycin concentration increased, resulting in full or partial inhibition of shoot regeneration. A significant or nonsignificant negative correlation occurred between kanamycin concentration and explant fresh weight over the time. Cefotaxime had little effect on regeneration potential. Growth of Agrobacterium cells was controlled at a concentration of 400 mg·L−1 cefotaxime in explants, and maximum putative transgenic shoot regeneration in explants of hypocotyls (44.88%) and cotyledons (36.29%) was obtained on MS (Murashige and Skoog)-selective shoot regeneration medium. Polymerase chain reaction analysis of genomic DNA using specifically designed primers detected the presence of the cryIAa and npt-II genes in kanamycin-resistant broccoli plantlets. Of five randomly selected putative transgenic shoots, three were positive for presence/integration of cryIAa and npt-II genes during T-DNA transfer and integration into the plant genome. Kanamycin and cefotaxime act as effective selective agents during genetic transformation pertaining to antibiotic sensitivity on cultured tissues of broccoli.